A systems approach using Diversity Outbred mice distinguishes the cardiovascular effects and genetics of circulating GDF11 from those of its homolog, myostatin.

Growth differentiation factor 11 (GDF11) is a member of the TGF-ß protein family that has been implicated in the development of cardiac hypertrophy. While some studies have suggested that systemic GDF11 protects against cardiomyocyte enlargement and left ventricular wall thickening, there remains uncertainty about the true impact of GDF11 and whether its purported effects are actually attributable to its homolog myostatin. This study was conducted to resolve the statistical and genetic relationships among GDF11, myostatin, and cardiac hypertrophy in a mouse model of human genetics, the Diversity Outbred (DO) stock. In the DO population, serum GDF11 concentrations positively correlated with cardiomyocyte cross-sectional area, while circulating myostatin levels were negatively correlated with body weight, heart weight, and left ventricular wall thickness and mass. Genetic analyses revealed that serum GDF11 concentrations are modestly heritable (0.23) and identified a suggestive peak on murine chromosome 3 in close proximity to the gene Hey1, a transcriptional repressor. Bioinformatic analyses located putative binding sites for the HEY1 protein upstream of the Gdf11 gene in the mouse and human genomes. In contrast, serum myostatin concentrations were more heritable (0.57) than GDF11 concentrations, and mapping identified a significant locus near the gene FoxO1, which has binding motifs within the promoter regions of human and mouse myostatin genes. Together, these findings more precisely define the independent cardiovascular effects of GDF11 and myostatin, as well as their distinct regulatory pathways. Hey1 is a compelling candidate for the regulation of GDF11 and will be further evaluated in future studies.

The Causal Effects of Blood Iron and Copper on Lipid Metabolism Diseases: Evidence from Phenome-Wide Mendelian Randomization Study.

Blood levels of iron and copper, even within their normal ranges, have been associated with a wide range of clinical outcomes. The available epidemiological evidence for these associations is often inconsistent and suffers from confounding and reverse causation. This study aims to examine the causal clinical effects of blood iron and copper with Mendelian randomization (MR) analyses. Genetic instruments for the blood levels of iron and copper were curated from existing genome-wide association studies. Candidate clinical outcomes were identified based on a phenome-wide association study (PheWAS) between these genetic instruments and a wide range of phenotypes in 310,999 unrelated individuals of European ancestry from the UK Biobank. All signals passing stringent correction for multiple testing were followed by MR analyses, with replication in independent data sources where possible. We found that genetically predicted higher blood levels of iron and copper are both associated with lower risks of iron deficiency anemia (odds ratio (OR) = 0.75, 95% confidence interval (CI): 0.67-0.85, p = 1.90 × 10-6 for iron; OR = 0.88, 95% CI: 0.78-0.98, p = 0.032 for copper), lipid metabolism disorders, and its two subcategories, hyperlipidemia (OR = 0.90, 95% CI: 0.85-0.96, p = 6.44 × 10-4; OR = 0.92, 95% CI: 0.87-0.98, p = 5.51 × 10-3) and hypercholesterolemia (OR = 0.90, 95% CI: 0.84-0.95, p = 5.34 × 10-4; OR = 0.93, 95% CI: 0.89-0.99, p = 0.022). Consistently, they are also associated with lower blood levels of total cholesterol and low-density lipoprotein cholesterol. Multiple sensitivity tests were applied to assess the presence of pleiotropy and the robustness of causal estimates. Regardless of the approaches, consistent evidence was obtained. Moreover, the unique clinical effects of each blood mineral were identified. Notably, genetically predicated higher blood iron is associated with an enhanced risk of varicose veins (OR = 1.28, 95% CI: 1.15-1.42, p = 4.34 × 10-6), while blood copper is positively associated with the risk of osteoarthrosis (OR = 1.07, 95% CI: 1.02-1.13, p = 0.010). Sex-stratified MR analysis further revealed some degree of sex differences in their clinical effects. Our comparative PheWAS-MR study of iron and copper comprehensively characterized their shared and unique clinical effects, highlighting their potential causal roles in hyperlipidemia and hypercholesterolemia. Given the modifiable nature of blood mineral status and the potential for clinical intervention, these findings warrant further investigation.

Genomic Characterization of the Zinc Transcriptional Regulatory Element Reveals Potential Functional Roles of ZNF658.

The zinc transcriptional regulatory element (ZTRE) is a newly reported binding motif for human zinc finger protein ZNF658, which alters gene expression in response to cellular zinc. The ZTRE has two nucleotide components-the palindromic flanking pairs and the bridging "N" bases between these flanks that range in number from 0 to 100. There are 12 pairs of ZTRE flanks (designated A-L). Three thousand five hundred twenty-five genes contain one or more ZTREs - 1000 to + 200 bp from their transcriptional start site (TSS). ZTRE-E is observed at a greater frequency, and ZTRE containing 25 bridging bases are less frequent, within - 200 bp from the TSS. The genes with ZTREs in this range are enriched in processes that may compensate zinc deficiency, while other genes with ZTREs outside this range are enriched in transcriptional activation processes. The division of ZTREs into two groups may imply a dual role of ZNF658, similar to the homologous yeast protein Zap1, via binding to low or high affinity sequences dependent upon cellular zinc. The KLF/Sp1-family binding motif is prevalent within the ZTRE "N" bridging bases, suggesting ZNF658 may compete with Sp1-like transactivators to suppress transcription.

MiRNA-target interactions in osteogenic signaling pathways involving zinc via the metal regulatory element.

Adequate zinc nutriture is necessary for normal bone growth and development, though the precise mechanisms for zinc-mediated bone growth remain poorly defined. A key transcription factor activated by zinc is metal response element-binding transcription factor 1 (MTF-1), which binds to the metal regulatory element (MRE). We hypothesize that MREs will be found upstream of miRNA genes as well as miRNA target genes in the following bone growth and development signaling pathways: TGF-ß, MAPK, and Wnt. A Bioconductor-based workflow in R was designed to identify interactions between MREs, miRNAs, and target genes. MRE sequences were found upstream from 64 mature miRNAs that interact with 213 genes which have MRE sequences in their own promoter regions. MAPK1 exhibited the most miRNA-target interactions (MTIs) in the TGF-ß and MAPK signaling pathways; CCND2 exhibited the most interactions in the Wnt signaling pathway. Hsa-miR-124-3p exhibited the most MTIs in the TGF-ß and MAPK signaling pathways; hsa-miR-20b-5p exhibited the most MTIs in the Wnt signaling pathway. MYC and hsa-miR-34a-5p were shared between all three signaling pathways, also forming an MTI unit. JUN exhibited the most protein-protein interactions, followed by MAPK8. These in silico data support the hypothesis that intracellular zinc status plays a role in osteogenesis through the transcriptional regulation of miRNA genes via the zinc/MTF-1/MRE complex.

Bioinformatic analysis of the metal response element and zinc-dependent gene regulation via the metal response element-binding transcription factor 1 in Caco-2 cells.

The purpose of this study was to determine the correlation between the position or number of metal regulatory elements (MREs) near gene transcriptional or translational start sites, and the strength of metal response element-binding transcription factor 1 (MTF-1) regulation. A secondary analysis was performed in silico on published results measuring the effects of Zn and MTF-1 on transcriptional regulation of genes (n = 120) in the Caco-2 cell line. MRE sequence variations throughout the human genome were sorted using a position weight matrix. Three null hypotheses (H0) were tested: (1) there is no correlation between the number of MREs and MTF-1 transcriptional strength, (2) there is no correlation between the distance of the MRE upstream from the transcriptional start site (TSS) and MTF-1 transcriptional strength, and (3) there is no correlation between the distance of the MRE downstream from the translational start site (TrSS) and MTF-1 transcriptional strength. Spearman correlation was used to test for significance (p < 0.05). From our results we rejected the first H0; we observed a significant correlation between the total number of MRE sequences - 7Kbp upstream from the TSS, within the 5' untranslated region, and + 1Kbp downstream from the TrSS, versus the strength of MTF-1 regulation (r = 0.202; p = 0.027). The second and third H0 were accepted. These results expand our understanding of the role of the MRE in Zn-dependent gene regulation. The data indicate that Zn influences the transcriptional control of gene expression beyond maintaining intracellular Zn homeostasis.

Zinc Supplementation Does Not Alter Indicators of Insulin Secretion and Sensitivity in Black and White Female Adolescents.

Background: Zinc is a micronutrient involved in the production of, and peripheral sensitivity to, pancreatic ß cell-derived insulin. To our knowledge, the effect of zinc supplementation on insulin outcomes, and potential risk of diabetes, in otherwise healthy children in the United States has not been investigated.Objective: The objective of this study was to determine the influence of zinc supplementation on insulin outcomes in black and white girls in the early stages of adolescence. A secondary objective was to determine relations between baseline zinc concentrations and insulin outcomes.Methods: Healthy black and white girls aged 9-11 y were randomly assigned to daily supplementation of zinc (9 mg elemental Zn/d; n = 75; blacks: n = 35) or placebo (n = 72; blacks: n = 32) for 4 wk. Fasting serum insulin, glucose, and C-peptide were assessed at baseline and at 4 wk. C-peptide and glucose values were used to calculate the computer model-derived homeostatic model assessment of insulin resistance (HOMA2-IR). Changes in outcome measures were compared by using repeated-measures, mixed-model ANOVA.Results: Baseline plasma zinc was not correlated with C-peptide (r = -0.07), insulin (r = -0.06), or HOMA2-IR (r = -0.09) (all P > 0.05) after controlling for race and age. Treatment × time interactions for C-peptide and HOMA2-IR were not significant (both P > 0.05). Although the treatment × race × time interactions for C-peptide and HOMA2-IR were not significant (both P = 0.08), black girls who received the placebo experienced slight increases in C-peptide (15.7%) and HOMA2-IR (17.7%) (P = 0.06).Conclusions: Four weeks of zinc supplementation had no effect on insulin outcomes in healthy black and white early-adolescent girls, although C-peptide and HOMA2-IR tended to increase in black girls who received placebo. Additional trials that are appropriately powered should further explore the effect of zinc on markers of diabetes risk, and whether race affects this relation. This trial was registered at clinicaltrials.gov as NCT01892098.

Zinc Supplementation Increases Procollagen Type 1 Amino-Terminal Propeptide in Premenarcheal Girls: A Randomized Controlled Trial.

BACKGROUND: Data have shown that healthy children and adolescents have an inadequate intake of zinc, an essential nutrient for growth. It is unclear whether zinc supplementation can enhance bone health during this rapid period of growth and development. OBJECTIVE: The primary aim of this study was to determine the effect of zinc supplementation on biochemical markers of bone turnover and growth in girls entering the early stages of puberty. The secondary aim was to test moderation by race, body mass index (BMI) classification, and plasma zinc status at baseline. METHODS: One hundred forty seven girls aged 9-11 y (46% black) were randomly assigned to a daily oral zinc tablet (9 mg elemental zinc; n = 75) or an identical placebo (n = 72) for 4 wk. Fasting plasma zinc, procollagen type 1 amino-terminal propeptide (P1NP; a bone formation marker), carboxy-terminal telopeptide region of type 1 collagen (ICTP; a bone resorption marker), and insulin-like growth factor I (IGF-I) were assessed at baseline and post-test. Additional markers of bone formation (osteocalcin) and resorption (urinary pyridinoline and deoxypyridinoline) were also measured. RESULTS: Four weeks of zinc supplementation increased plasma zinc concentrations compared with placebo [mean change, 1.8 µmol/L (95% CI: 1.0, 2.6) compared with 0.2 µmol/L (95% CI: -0.3, 0.7); P < 0.01]. Zinc supplementation also increased serum P1NP concentrations compared with placebo [mean change, 23.8 µmol/L (95% CI: -14.9, 62.5) compared with -31.0 µmol/L (95% CI: -66.4, 4.2); P = 0.04). There was no effect from zinc supplementation on osteocalcin, ICTP, pyridinoline, deoxypyridinoline, or IGF-I. There was no moderation by race, BMI classification, or plasma zinc status at baseline. CONCLUSIONS: Our data suggest that 4 wk of zinc supplementation increases bone formation in premenarcheal girls. Further studies are needed to determine whether supplemental zinc can improve childhood bone strength. This trial was registered at clinicaltrials.gov as NCT01892098.

Zinc affects miR-548n, SMAD4, SMAD5 expression in HepG2 hepatocyte and HEp-2 lung cell lines.

MicroRNAs affect disease progression and nutrient status. miR-548n increased 57 % in Zn supplemented plasma from adolescent females (ages 9 to 13 years). The purpose of this study was to determine the effects of Zn concentration in cell culture on the expression of miR-548n, SMAD4 and SMAD5 in hepatocyte (HepG2) and lung epithelium (HEp-2) cell lines. Cells were incubated for 48 h in media containing 10 % Chelex 100-treated FBS (0 µM Zn), or with 15 or 50 µM Zn, before isolation of total RNA and cDNA. Expression of miR-548n, SMAD4 and SMAD5 was measured by qPCR. The ΔΔCT method was used to calculate the fold-change, and 15 µM expression levels were used as reference values. HepG2 miR-548n expression decreased 5-fold, and SMAD4 expression increased 4-fold in the absence of Zn, while HEp-2 miR-548n expression increased 10.5-fold, and SMAD5 expression increased 20-fold in the absence of Zn. HEp-2 miR-548n expression increased 23-fold, while SMAD4 expression decreased twofold, in 50 µM Zn-treated cells. However, SMAD4 and SMAD5 expression was not correlated. These data indicate that miR-548n expression is in part regulated by Zn in a cell-specific manner. SMAD4 and SMAD5 are genes in the TGF-ß/BMP signaling pathway, and SMAD5 is a putative target for miR-548n; Zn participates in regulating this pathway through controlling SMAD4 and SMAD5 expression. However, SMAD5 expression may be more sensitive to Zn than to miR-548n since SMAD5 expression was not inversely correlated with miR-548n expression.

Genetic analysis of tissue glutathione concentrations and redox balance.

Glutathione redox balance-defined as the ratio GSH/GSSG-is a critical regulator of cellular redox state, and declines in this ratio are closely associated with oxidative stress and disease. However, little is known about the impact of genetic variation on this trait. Previous mouse studies suggest that tissue GSH/GSSG is regulated by genetic background and is therefore heritable. In this study, we measured glutathione concentrations and GSH/GSSG in liver and kidney of 30 genetically diverse inbred mouse strains. Genetic background caused an approximately threefold difference in hepatic and renal GSH/GSSG between the most disparate strains. Haplotype association mapping determined the loci associated with hepatic and renal glutathione phenotypes. We narrowed the number of significant loci by focusing on those located within protein-coding genes, which we now consider to be candidate genes for glutathione homeostasis. No candidate genes were associated with both hepatic and renal GSH/GSSG, suggesting that genetic regulation of GSH/GSSG occurs predominantly in a tissue-specific manner. This is the first quantitative trait locus study to examine the genetic regulation of glutathione concentrations and redox balance in mammals. We identified novel candidate genes that have the potential to redefine our knowledge of redox biochemistry and its regulation and inform future therapeutic applications.

Dietary zinc depletion and repletion affects plasma proteins: an analysis of the plasma proteome.

Zinc (Zn) deficiency is a problem world-wide. Current methods for assessing Zn status are limited to measuring plasma or serum Zn within populations suspected of deficiency. Despite the high prevalence of Zn deficiency in the human population there are no methods currently available for sensitively assessing Zn status among individuals. The purpose of this research was to utilize a proteomic approach using two-dimensional gel electrophoresis (2DE) and mass spectrometry to identify protein biomarkers that were sensitive to changes in dietary Zn levels in humans. Proteomic analysis was performed in human plasma samples (n = 6) obtained from healthy adult male subjects that completed a dietary Zn depletion/repletion protocol, current dietary zinc intake has a greater effect on fractional zinc absorption than does longer term zinc consumption in healthy adult men. Chung et al. (Am J Clin Nutr 87 (5):1224-1229, 2008). After a 13 day Zn acclimatization period where subjects consumed a Zn-adequate diet, the male subjects consumed a marginal Zn-depleted diet for 42 days followed by consumption of a Zn-repleted diet for 28 days. The samples at baseline, end of depletion and end of repletion were pre-fractionated through immuno-affinity columns to remove 14 highly abundant proteins, and each fraction separated by 2DE. Following staining by colloidal Coomassie blue and densitometric analysis, three proteins were identified by mass spectrometry as affected by changes in dietary Zn. Fibrin ß and chain E, fragment double D were observed in the plasma protein fraction that remained bound to the immunoaffinity column. An unnamed protein that was related to immunoglobulins was observed in the immunodepleted plasma fraction. Fibrin ß increased two-fold following the Zn depletion period and decreased to baseline values following the Zn repletion period; this protein may serve as a viable biomarker for Zn status in the future.

Marginal maternal zinc deficiency in lactating mice reduces secretory capacity and alters milk composition.

Dietary analysis predicts that marginal Zn deficiency is common in women of reproductive age. The lack of reliable biomarkers limits the capacity to assess Zn status and consequently understand effects of maternal Zn deficiency. We determined effects of marginal maternal Zn deficiency on mammary gland function, milk secretion, and milk composition in mice. Mice (n = 12/diet) were fed marginal (ZD; 15 mg Zn/kg diet) or adequate (ZA; 30 mg Zn/kg diet) Zn diets for 30 d prior to conception through mid-lactation. Mice fed the ZD had a higher plasma Zn concentration (~20%; P < 0.05) but lower milk Zn concentration (~15%; P < 0.05) compared with mice fed the ZA. ZnT2 abundance was higher (P < 0.05) in mice fed the ZD compared with mice fed the ZA; no effect on ZnT4 abundance was detected. The Zn concentration of mammary gland mitochondria tended to be ~40% greater in mice fed ZD (P = 0.07); this was associated with apoptosis and lower milk secretion (~80%; P < 0.01). Total milk protein was ~25% higher (P < 0.05), although the abundance of the major milk proteins (caseins and whey acidic protein) was lower (P < 0.05) in mice fed the ZD. Proteomic analysis of milk proteins revealed an increase (P < 0.05) in four proteins in mice fed the ZD. These findings illustrate that marginal maternal Zn deficiency compromises mammary gland function and milk secretion and alters milk composition. This suggests that lactating women who consume inadequate Zn may not produce and/or secrete an adequate amount of high quality milk to provide optimal nutrition to their developing infant.

Can the soil bacterium Cupriavidus necator sense ZnO nanomaterials and aqueous Zn²âº differentially?

Synthetic metal oxide nanomaterials exert toxicity via two general mechanisms: release of free ions at concentrations which exert toxic effects upon the target cell, or specific surface-mediated physicochemical processes leading to the formation of hydroxyl free radicals and other reactive oxygen species which act to disrupt cell membranes and organelles. From a regulatory standpoint this presents a potential problem since it is not trivial to detect free metal ions in the presence of nanoparticles in biological or natural media. This makes efforts to identify the route of uptake difficult. Although in vitro studies of zinc oxide nanoparticles suggest that toxicity to the soil bacterium Cupriavidus necator is exerted in a similar manner to zinc acetate, we found no free Zn ion is associated with nanoparticle suspensions. The proteome of cells subjected to equal concentrations of either the nanoparticle or zinc acetate suggest that the mode of toxicity is quite different for the two forms of Zn, with a number of membrane-associated proteins up-expressed in response to nanoparticle exposure. Our data suggests that nanoparticles act to interrupt cell membranes thereby causing cell death rather than exerting a strictly toxic effect. We also identify potentially useful genes to serve as biomarkers of membrane disruption in toxicogenomic studies with nanoparticles or to engineer biosensor organisms.

Proteomic analysis and differential expression in protein extracted from chicken with a varying growth rate and water-holding capacity.

Chickens from a randomly bred genetic line were segregated into high and low growth rates and high and low water-holding capacities (WHCs). The objective of this study was to identify protein markers associated with slow and fast growth rates and low and high WHCs from water-soluble protein (WSP) and crude myofibrillar protein (CMP) extracts of chicken breast muscle. Proteins were fractionated using two-dimensional electrophoresis, and a total of 22 protein spots were selected, excised, and analyzed by in-gel tryptic digestion and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Proteins expressed in extracts from slow and fast growth rates and low and high WHCs included metabolic enzymes, such as creatine kinase, pyruvate kinase, triosephosphate isomerase, and ubiqitin; housekeeping proteins, such as heat shock protein; contractile proteins, such as myosin heavy chain; actin; and also MHC isoforms and actin isoforms. The mass spectra of 20 protein spots significantly matched (protein score >83; P < 0.05) an online database. In CMP, there were unique proteins that were present only in the fast-growth population: gi|118099530 , gi|20664362 , gi|71895043 , gi|114794125 , gi|297343122 , and gi|71895043 . This information identified protein markers associated with growth rate and water holding capacity. Some of those protein markers could be added to the chicken database.

Analysis of the liver soluble proteome from bull terriers affected with inherited lethal acrodermatitis.

Lethal acrodermatitis (LAD) is a genetic disease affecting bull terrier dogs. The phenotype is similar to that for acrodermatitis enteropathica in humans, but is currently without treatment. The purpose of the research presented here is to determine the biochemical defects associated with LAD using proteomic methodologies. Two affected (male and female) and one unaffected (male) bull terrier pups were euthanized at 14 weeks of age, their livers dissected and prepared for two-dimensional gel electrophoresis (2DE) and densitometry. Approximately 200 protein spots were observed. The density of the spots within each gel was normalized to the total spot volume of the gel; only those soluble liver protein spots that were consistently different in both of the livers of the affected pups compared to the unaffected pup were excised manually and submitted for MALDI mass spectrometry. Thirteen proteins were identified as differentially expressed in the affected, compared to the unaffected, pups. The proteins were involved in numerous cellular physiological functions, including chaperones, calcium binding, and energy metabolism, as well as being associated with the inflammatory response. Of note were haptoglobin, glutamine synthetase, prohibitin and keratin 10 which exhibited at least a fourfold level of differential expression. These data represent the first proteomic analysis of this mutation. The differentially expressed proteins that were identified may be key in understanding the etiology of LAD, and may lead to diagnostic tools for its identification within the bull terrier population.

Consumption of a moderately Zn-deficient and Zn-supplemented diet affects soluble protein expression in rat soleus muscle.

Zinc deficiency negatively affects muscle function, but there are limited biochemical data identifying the cause of this reduction in function. The objective of the present study was to identify soluble proteins in rat soleus muscle that were responsive to different levels of dietary zinc. Rats (n=21) were fed diets containing three concentrations of zinc: 5, 30 and 200 ppm for 42 days. There was no difference in body weights of the rats consuming the 5-ppm zinc diet compared to the rats consuming the 30- or 200-ppm zinc diets; however, bone zinc levels were significantly decreased in the 5-ppm dietary zinc group. Individual soluble protein fractions were isolated from these muscles and the samples were prepared for two-dimensional polyacrylamide gel electrophoresis. The expression levels of four proteins were significantly depressed by dietary Zn depletion and supplementation, S-glutathiolated carbonic anhydrase, myosin light polypeptide 3, heat shock protein 20 and heart fatty acid binding protein. This is the first report that indicates that both Zn depletion and supplementation result in protein expression profiles that may negatively affect skeletal muscle function. These results indicate that there are specific signaling pathways that require proper Zn nutriture for maintaining optimal muscle function and suggest that the consumption of pharmacologic doses of Zn may be detrimental to muscle function.

The effects of quercetin on SW480 human colon carcinoma cells: a proteomic study.

BACKGROUND: High fruit and vegetable intake is known to reduce the risk of colon cancer. To improve understanding of this phenomenon the action of different phytochemicals on colon cells has been examined. One such compound is quercetin that belongs to the group known as flavonoids. The purpose of this study was to determine the influence of quercetin on the proteome of the SW480 human colon adenocarcinoma cell line, specifically to identify proteins that could be the molecular targets of quercetin in its amelioration of the progression of colon cancer. To this end, two-dimensional gel electrophoresis and mass spectrometry were used to identify proteins that underwent a change in expression following treatment of the cells with 20 muM quercetin. This could elucidate how quercetin may reduce the progression of colon cancer. RESULTS: Quercetin treatment of the SW480 human colon cancer cells was found to result in the decreased expression of three proteins and the increased expression of one protein. The identified proteins with decreased expression were type II cytoskeletal 8 keratin and NADH dehydrogenase Fe-S protein 3. The other protein with decreased expression was not identified. The protein with increased expression belonged to the annexin family. CONCLUSION: Several proteins were determined to have altered expression following treatment with quercetin. Such changes in the levels of these particular proteins could underlie the chemo-protective action of quercetin towards colon cancer.

Expression of P2X6, a purinergic receptor subunit, is affected by dietary zinc deficiency in rat hippocampus.

The purpose of these experiments was to determine whether dietary zinc depletion affected protein expression in the hippocampus. Eleven weanling Sprague-Dawley male rats (21 d) were fed the AIN-93G diet containing 1.5 ppm zinc and supplemented with 30 ppm of zinc in the drinking water. After 1 wk, the rats were randomly divided into three groups: control (n=3), pair fed (n=3), and zinc restricted (n=5). All groups consumed the same diet. The zinc-restricted group consumed water containing no zinc. The rats were sacrificed 3 wk later. Chelatable zinc levels in the hippocampus, as measured by N-(6-methoxy-8-quinolyl)-para-toluenesulfonamide (TSQ) staining, were significantly reduced in the zinc-restricted group. Analysis of hippocampal protein expression by two-dimensional electrophoresis (2DE) revealed increased expression of the P2X6 purinergic receptor in the zinc-restricted rats, as determined by MALDI mass spectrometry (MS) and database analysis. The data provided evidence for the dual effects of dietary zinc deficiency on the hippocampus, reducing ionic zinc levels and stimulating protein expression. The role the P2X6 receptor plays in the physiological response of the hippocampus to zinc depletion remains to be determined.

Zinc uptake into MCF-10A cells is inhibited by cholesterol depletion.

The mechanism for cellular Zn uptake was investigated by depleting cell cholesterol levels, a treatment that disrupts lipid rafts/caveolae-dependent processes and inhibits coated-pit budding. Incubation of MCF-10A human breast epithelial cells with hydroxypropyl-beta-cyclodextrin significantly lowered cell cholesterol levels and significantly inhibited cellular zinc uptake measured at 10 min, but had no effect on 2-deoxyglucose uptake. Replacing potassium for sodium in the uptake buffer significantly stimulated Zn uptake by 20%. The effects of potassium depletion and chlorpromazine on Zn uptake were investigated to determine the contribution of coated-pit endocytosis. Potassium depletion following hypotonic shock significantly inhibited Zn uptake into MCF-10A cells approximately 15%. Chlorpromazine at 20 microg/ml inhibited uptake approximately 30%. The data support the hypothesis that Zn uptake into MCF-10A cells involves lipid rafts/caveolae. The relatively mild effects of potassium depletion and chlorpromazine suggest that a small portion of Zn uptake may require coated pit endocytosis.