A systems-approach to NAD+ restoration.

A decline in NAD+ is a feature of ageing and may play a causal role in the process. NAD+ plays a pivotal role in myriad processes important in cellular metabolism and is a cosubstrate for enzymes that play key roles in pathways that modify ageing. Thus, interventions that increase NAD+ may slow aspects of the ageing trajectory and there is great interest in pharmacological NAD+ restoration. Dietary supplementation with NAD+ precursors, particularly nicotinamide riboside, has increased NAD+ levels in several human intervention studies and arguably been the most robust approach to date. However, consistency and reliability of such approaches to increase NAD+, and also impact on markers of efficacy to slow or reverse features of ageing, has been inconsistent. We argue that a major element of this variability may arise from the use of single-target approaches that do not consider the underlying biological complexity leading to NAD+ decline. Thus, a systems approach - targeting multiple key nodes in the NAD+ interactome - is likely to be more efficacious and reliable.

Impact of In Utero Folate Exposure on DNA Methylation and Its Potential Relevance for Later-Life Health-Evidence from Mouse Models Translated to Human Cohorts.

SCOPE: Persistent DNA methylation changes may mediate effects of early-life exposures on later-life health. Human lifespan is challenging for prospective studies, therefore data from longitudinal studies are limited. Projecting data from mouse models of early-life exposure to human studies offers a tool to address this challenge. METHODS AND RESULTS: C57BL/6J mice were fed low/normal folate diets before and during pregnancy and lactation. Genome-wide promoter methylation was measured in male offspring livers at 17.5 days gestation and 28 weeks. Eight promoters were concurrently hypermethylated by folate depletion in fetuses and adults (>1.10 fold-change; p < 0.05). Processes/pathways potentially influenced by global changes, and function of these eight genes, suggest neurocognitive effects. Human observational and randomized controlled trial data were interrogated for translation. Methylation at birth was inversely associated with maternal plasma folate in six genes (-1.15% to -0.16% per nmol L-1 ; p < 0.05), while maternal folic acid supplementation was associated with differential methylation of four genes in adulthood. Three CpGs were persistently hypermethylated with lower maternal folate (p = 0.04). CONCLUSION: Some persistent folate-induced methylation changes in mice are mirrored in humans. This demonstrates utility of mouse data in identifying human loci for interrogation as biomarkers of later-life health.

Antileukemic Effect of Palladium Nanoparticles Mediated by White Tea (Camellia sinensis) Extract In Vitro and in WEHI-3B-Induced Leukemia In Vivo.

This study investigated the in vivo antileukemic activity of palladium nanoparticles (Pd@W.tea-NPs) mediated by white tea extract in a murine model. The cell viability effect of Pd@W.tea-NPs, "blank" Pd nanoparticles, and white tea extract alone was determined in murine leukemia WEHI-3B cells and normal mouse fibroblasts (3T3 cells). Apoptotic and cell cycle arrest effects of Pd@W.tea-NPs in WEHI-3B cells were evaluated. The effects of Pd@W.tea-NPs administered orally to leukemic mice at 50 and 100 mg/kg daily over 28 days were evaluated. Pd@W.tea-NPs reduced the viability of WHEI-3B cells with IC50 7.55 µg/ml at 72 h. Blank Pd nanoparticles and white tea extract alone had smaller effects on WHEI-3B viability and on normal fibroblasts. Pd@W.tea-NPs increased the proportion of Annexin V-positive WHEI-3B cells and induced G2/M cell cycle arrest. Leukemic cells in the spleen were reduced by Pd@W.tea-NPs with an increase in Bax/Bcl-2 and cytochrome-C protein and mRNA levels indicating the activation of the mitochondrial apoptotic pathway. These effects replicated the effects of ATRA and were not observed using blank Pd nanoparticles. Pd@W.tea-NPs afford therapeutic efficacy against leukemia likely to pivot on activation of the mitochondrial pathway of apoptotic signaling and hence appear attractive potential candidates for development as a novel anticancer agent.

Changes in Zn homeostasis during long term culture of primary endothelial cells and effects of Zn on endothelial cell senescence.

Endothelial cell senescence and Zn nutritional status influence cardiovascular disease. The influence of Zn appears dichotomous, hence it is imperative to understand the relationship with cellular senescence to improve knowledge about the molecular and cellular basis of the disease. Here we aimed to determine: 1) the impact of chronic exposure to a moderately high dose of Zn on senescence of endothelial cells; 2) the changes in Zn homeostasis during the lifespan of primary cultured endothelial cells; and 3) the susceptibility of proliferating and senescent endothelial cells to cell death after short term exposure to increasing doses of Zn and of the Zn chelator TPEN. Chronic exposure to Zn accelerated senescence and untreated cells at later passages, where doubling time had increased, displayed relocation of labile Zn and altered expression of genes involved in the response to Zn toxicity, including SLC30A1, SLC39A6, SLC30A5, SLC30A10 and metallothioneins, indicating that senescent cells have altered zinc homeostasis. Most Zn-dependent genes that were expressed differently between early and late passages were correlated with changes in the expression of anti-apoptotic genes. Short-term treatment with a high dose of Zn leads to cell death, but only in the population of cells at both earlier and later passages that had already entered senescence. In contrast, Zn depletion led to death of cells at earlier but not later passages, which suggests that there are sub-populations of senescent cells that are resistant to Zn depletion. This resistant senescent cell population may accumulate under conditions of Zn deficiency and contribute to vascular pathology.

Selenium alters miRNA profile in an intestinal cell line: evidence that miR-185 regulates expression of GPX2 and SEPSH2.

SCOPE: Intake of the essential micronutrient selenium (Se) has health implications. This work addressed whether some effects of Se on gene expression are exerted through microRNAs (miRNA). METHODS AND RESULTS: Human colon adenocarcinoma cells (Caco-2) were grown in Se-deficient or Se-adequate medium for 72 h. RNA was extracted and subjected to analysis of 737 miRNA using microarray technology. One hundred and forty-five miRNA were found to be expressed in Caco-2 cells. Twelve miRNA showed altered expression after Se depletion: miR-625, miR-492, miR-373*, miR-22, miR-532-5p, miR-106b, miR-30b, miR-185, miR-203, miR1308, miR-28-5p, miR-10b. These changes were validated by quantitative real-time PCR (RT-qPCR). Transcriptomic analysis showed that Se depletion altered expression of 50 genes including selenoproteins GPX1, SELW, GPX3, SEPN1, SELK, SEPSH2 and GPX4. Pathway analysis identified arachidonic acid metabolism, glutathione metabolism, oxidative stress, positive acute phase response proteins and respiration of mitochondria as Se-sensitive pathways. Bioinformatic analysis identified 13 transcripts as targets for the Se-sensitive miRNA; three were predicted to be recognised by miR-185. Silencing of miR-185 increased GPX2 and SEPSH2 expression. CONCLUSIONS: We propose that miR-185 plays a role in up-regulation of GPX2 and SEPHS2 expression. In the case of SEPHS2 this may contribute to maintaining selenoprotein synthesis. The data indicate that micronutrient supply can regulate the cell miRNA expression profile.

Altered expression of ZnT10 in Alzheimer's disease brain.

There is an increasing body of evidence suggesting that metal homeostasis is dysregulated in the pathology of Alzheimer's disease (AD). Although expression levels of several transporters belonging the SLC30 family, which comprises predominantly zinc transporters, have been studied in the AD brain, SLC30A10 (ZnT10) has not been studied in this context. To determine if dysregulated expression of ZnT10, which may transport both Zn and Mn, could be a factor that contributes to AD, we investigated if there were differences in ZnT10 mRNA levels in specimens of frontal cortex from AD patients and controls and also if brain tissue from the APP/PS1 transgenic (Tg) mouse model showed abnormal levels of ZnT10 mRNA expression. Our results show that ZnT10 is significantly (P<0.01) decreased in the frontal cortex in AD. Furthermore, we observed a significant decrease in ZnT10 mRNA levels in the APP/PS1-Tg mice compared with wild-type controls (P<0.01). Our results suggest that this dysregulation in ZnT10 could further contribute to disease progression.

Honeybees and cell lines as models of DNA methylation and aging in response to diet.

DNA methylation patterns change as individuals grow older, and DNA methylation appears susceptible to modification by the diet. Thus DNA methylation may be a mechanism through which diet can affect aging and longevity. We propose that effects on DNA methylation also contribute to the extension in lifespan observed in response to dietary restriction. Relationships between diet-induced changes in DNA methylation and parallel effects on aging and/or lifespan could, of course, be purely associative. Proof of these ideas requires experimental model systems in which it is possible to manipulate genome methylation status and to measure effects on aging and/or lifespan. Commonly-used short-lived and genetically-malleable metazoan species, such as Caenorhabditis elegans and Drosophila, are not suitable for such studies; the C. elegans genome is not methylated, and DNA methylation in Drosophila is dissimilar from mammalian DNA methylation, occurring at cytosines at sites other than in CpG sequences. The honeybee provides a potentially unique and tractable model for such studies. Female larval development into the long-lived queen phenotype or short-lived worker is determined purely by diet (royal jelly) through an effect on DNA methylation, and honeybee DNA methylation mirrors that of the mammalian genome. Mammalian cell lines and biochemical approaches offer complementary tools to address specific components of hypotheses relating to effects of diet on aging through DNA methylation in a more targeted manner. Our studies using mammalian cell lines are revealing effects of Sirt1 on DNA methylation, and indicate that Sirt1 and resveratrol affect the expression of different sets of genes.

Polymorphisms in genes involved in the metabolism and transport of soy isoflavones affect the urinary metabolite profile in premenopausal women following consumption of a commercial soy supplement as a single bolus dose.

SCOPE: Genetic variation in relevant enzymes and transporters may contribute to discordant observations concerning health outcomes of dietary isoflavone consumption, so we examined the association of the UGT1A1*28 promoter polymorphism and of other SNPs with isoflavone metabolites in urine. METHODS AND RESULTS: We genotyped prospectively for polymorphisms in UGT1A1 (UGT1A1*28), LPH (666G>A), CBG (1368T>A), ABCG2 (421C>A), and ABCC2 (1249G>A) to select 100 women (18-50 years) to receive a commercial soy supplement as a single dose and collect all urine over 24 h for analysis by RP-HPLC. We observed large differences in isoflavone recovery (mean 39%, eightfold variation) and metabolites. Glucuronides were the major metabolites (72% of total). UGT1A1*28 was associated only with percentage of glycitein as sulphate (positive; p = 0.046), but excluding five participants with both minor alleles of CBG and ABCG2 uncovered additional associations with percentage of glycitein as glucuronide (negative; p = 0.028), combined isoflavones as sulphate (positive; p = 0.035) and sulphate-to-glucuronide ratio for combined isoflavones (positive; p = 0.036). CBG1368T>A, ABCG2 421C>A, and ABCC2 1249G>A were also associated with differences in isoflavone metabolites in urine. CONCLUSION: Genetic variation in UGT1A1, CBG, ABCG2, and ABCC2 influences isoflavone metabolism so may affect benefits of dietary consumption.

The potential role of epigenetic responses to diet in ageing.

Epigenetic changes may be causal in the ageing process and may be influenced by diet, providing opportunities to improve health in later life. The aim of this review is to provide an overview of several areas of research relevant to this topic and to explore a hypothesis relating to a possible role of epigenetic effects, mediated by sirtuin 1, in the beneficial effects of dietary restriction, including increased lifespan. Epigenetic features of ageing include changes in DNA methylation, both globally and at specific loci, which differ between individuals. A major focus of research on dietary influences on epigenetic status has been on nutrition in utero, because the epigenome is probably particularly malleable during this life-course window and because epigenetic marking by early exposures is a compelling mechanism underlying effects on lifelong health. We explore the potential of diet during adulthood, including the practice of dietary restriction, to affect the epigenetic architecture. We report progress with respect to deriving data to support our hypothesis that sirtuin 1 may mediate some of the effects of dietary restriction through effects on DNA methylation and note observations that resveratrol affects DNA methylation and other epigenetic features. Disentangling cause and effect in the context of epigenetic change and ageing is a challenge and requires better understanding of the underlying mechanisms and also the development of more refined experimental tools to manipulate the epigenetic architecture, to facilitate hypothesis-driven research to elucidate these links and thus to exploit them to improve health across the full life-course through dietary measures.

Selenium in human health and disease.

This review covers current knowledge of selenium in the environment, dietary intakes, metabolism and status, functions in the body, thyroid hormone metabolism, antioxidant defense systems and oxidative metabolism, and the immune system. Selenium toxicity and links between deficiency and Keshan disease and Kashin-Beck disease are described. The relationships between selenium intake/status and various health outcomes, in particular gastrointestinal and prostate cancer, cardiovascular disease, diabetes, and male fertility, are reviewed, and recent developments in genetics of selenoproteins are outlined. The rationale behind current dietary reference intakes of selenium is explained, and examples of differences between countries and/or expert bodies are given. Throughout the review, gaps in knowledge and research requirements are identified. More research is needed to improve our understanding of selenium metabolism and requirements for optimal health. Functions of the majority of the selenoproteins await characterization, the mechanism of absorption has yet to be identified, measures of status need to be developed, and effects of genotype on metabolism require further investigation. The relationships between selenium intake/status and health, or risk of disease, are complex but require elucidation to inform clinical practice, to refine dietary recommendations, and to develop effective public health policies.

Glucuronidation of the soyabean isoflavones genistein and daidzein by human liver is related to levels of UGT1A1 and UGT1A9 activity and alters isoflavone response in the MCF-7 human breast cancer cell line.

The soyabean isoflavones genistein and daidzein, which may protect against some cancers, cardiovascular disease and bone mineral loss, undergo substantial Phase 2 metabolism, predominantly glucuronidation. We observed a correlation between rates of metabolism of marker substrates of specific UGTs and rates of glucuronidation of genistein and daidzein in vitro by a panel of human liver microsomes, demonstrating that UGT1A1 and UGT1A9, but not UGT1A4, make a major contribution to the metabolism of these isoflavones by human liver. These findings were substantiated by observations that recombinant human UGT1A1 and UGT1A9, but not UGT1A4, catalysed the production of the major glucuronides of both genistein and daidzein in vitro. Recombinant human UGT1A8 also metabolised both genistein and daidzein, whereas UGT1A6 was specific to genistein and UGTs 2B7 and 2B15 were inactive, or only marginally active, with either isoflavone as substrate. The intestinal isoform UGT1A10 metabolised either both isoflavones or genistein only, depending on the commercial supplier of the recombinant enzyme, possibly as a result of a difference in amino acid sequence, which we were unable to confirm. Daidzein (16 microM) increased cell death in the MCF-7 human breast cancer cell line and this effect was reversed by glucuronidation. In view of a well-characterised functional polymorphism in UGT1A1, these observations may have implications for inter-individual variability in the potential health-beneficial effects of isoflavone consumption.

Intracellular zinc homeostasis in leukocyte subsets is regulated by different expression of zinc exporters ZnT-1 to ZnT-9.

Intracellular zinc homeostasis is strictly regulated by zinc binding proteins and zinc transporters. In the present study, we quantified in a first global view the expression of all characterized human zinc exporters (hZnT-1-9) in different leukocyte subsets in response to zinc supplementation and depletion and analyzed their influence on alterations in the intracellular zinc concentration. We found that hZnT-1 is the most regulated zinc exporter. Furthermore, we discovered that hZnT-4 is localized in the plasma membrane similar to hZnT-1. hZnT-4 is most highly expressed in Molt-4, up-regulated after treatment with PHA and is responsible for the measured decrease of intracellular zinc content after high zinc exposure. In addition, we found that hZnT-5, hZnT-6, and hZnT-7 in Raji as well as hZnT-6 and hZnT-7 in THP-1 are up-regulated in response to cellular zinc depletion. Those zinc exporters are all localized in the Golgi network, and this type of regulation explains the observed zinc increase in both cell types after up-regulation of their expression during zinc deficiency and, subsequently, high zinc exposure. Furthermore, we detected, for the first time, the expression of hZnT-8 in peripheral blood lymphocytes, which varied strongly between individuals. While hZnT-2 was not detectable, hZnT-3 and hZnT-9 were expressed at low levels. Further on, the amount of expression was higher in primary cells than in cell lines. These data provide insight into the regulation of intracellular zinc homeostasis in cells of the immune system and may explain the variable effects of zinc deficiency on different leukocyte subsets.

Intestinal and placental zinc transport pathways.

Mammalian members of the cation diffusion facilitator (CDF) and zrt-, irt-like protein (ZIP) families of Zn transporters, initially identified in Saccharomyces cerevisiae and Arabidopsis thalania spp., have been cloned during the last 8 years and have been classified as families SLC30 and SLC39 respectively. The cloning of human Zn transporters ZnT-like transporter 1 (hZTL1)/ZnT5 (SLC30A5) and hZIP4 (SLC39A4) were major advances in the understanding of the molecular mechanisms of dietary Zn absorption. Both transporters are localised at the enterocyte apical membrane and are, therefore, potentially of fundamental importance in dietary Zn uptake. hZTL1 mediates Zn uptake when expressed in Xenopus laevis oocytes and hZIP4 is mutated in most cases of the inherited Zn deficiency disease acrodermatitis enteropathica. Localisation of hZTL1/ZnT5 at the apical membrane of the placental syncytiotrophoblast indicates a fundamental role in the transfer of Slc30 Zn to the foetus. Observations in rodent models indicate that in the intestine increased Zn availability increases expression of Zn transporters. Human intestinal Caco-2 cells show a similar response to increasing the Zn2+ concentration of the nutrient medium in relation to the expression of mRNA corresponding to several Zn transporters and that of ZnT1 (SLC30A1) and hZTL1/ZnT5 proteins. In the human placental cell line JAR, however, expression at the mRNA level of a number of Zn transporters is not modified by Zn availability, whilst ZnT1 and hZTL1/ZnT5 proteins are reduced under Zn-supplemented conditions. These differences between Caco-2 and JAR cells in Zn transporter gene responses to Zn supply may reflect the different extracellular Zn concentrations encountered by the corresponding cell types in vitro.

A novel zinc-regulated human zinc transporter, hZTL1, is localized to the enterocyte apical membrane.

Zinc is essential to a wide range of cellular processes; therefore, it is important to elucidate the molecular mechanisms of zinc homeostasis. To date, no zinc transporters expressed at the enterocyte apical membrane, and so essential to mammalian zinc homeostasis, have been discovered. We identified hZTL1 as a human expressed sequence tag with homology to the basolateral enterocyte zinc transporter ZnT1 and deduced the full-length cDNA sequence by PCR. The protein of 523 amino acids belongs to the cation diffusion facilitator family of membrane transporters. Unusually, the predicted topology comprises 12 rather than 6 transmembrane domains. ZTL1 mRNA was detected by reverse transcription-PCR in a range of mouse tissues. A Myc-tagged hZTL1 clone was expressed in transiently transfected polarized human intestinal Caco-2 cells at the apical membrane. Expression of hZTL1 mRNA in Caco-2 cells increased with zinc supplementation of the nutrient medium; however, in the placental cell line JAR hZTL1 appeared not to be regulated by zinc. Heterologous expression of hZTL1 in Xenopus laevis oocytes increased zinc uptake across the plasma membrane. The localization, regulatory properties, and function of hZTL1 indicate a role in regulating the absorption of dietary zinc across the apical enterocyte membrane.