Chiara Murgia

Diabetes-linked zinc transporter ZnT8 is a homodimeric protein expressed by distinct rodent endocrine cell types in the pancreas and other glands

BACKGROUND AND AIMS Zinc is abundant in pancreas, being required by endocrine islet cells for hormone secretion and by exocrine acinar cells as pancreatic juice component. ZnT8 is a member of the SLC30A family of zinc transporters whose overexpression in cultured pancreatic beta cells leads to increased insulin secretion in response to glucose, suggesting a possible role in regulating glycemia. ZnT8 was therefore proposed as a therapeutic target for diabetes, and recent genome-wide association studies identified polymorphisms in the ZNT8 gene conferring increased type 2 diabetes risk. METHODS AND RESULTS As limited information was available on the biochemical properties of ZnT8 and on its endogenous expression, we have raised a specific polyclonal antibody and immunostained protein extracts, cell lines and tissue sections. We show that ZnT8 forms a very stable dimer that requires biological membranes to properly assemble. We demonstrate localization of murine ZnT8 to the secretory granules in pancreatic beta and alpha islet cells. Moreover, we show that ZnT8 is also expressed in other secretory cell types, namely the cubical epithelium that lines thyroid follicles and the cortex of the adrenal gland, suggesting a more widespread role in endocrine secretion. CONCLUSION We provide novel insights into the features of the ZnT8 transporter, of special relevance in light of its proposed role as therapeutical target for diabetes treatment.

Labile zinc and zinc transporter ZnT4 in mast cell granules: Role in regulation of caspase activation and NF-KB translocation

The granules of mast cells and other inflammatory cells are known to be rich in zinc (Zn), a potent caspase inhibitor. The functions of granular Zn, its mechanism of uptake, and its relationship to caspase activation in apoptosis are unclear. The granules of a variety of mast cell types fluoresced intensely with the Zn-specific fluorophore Zinquin, and fluorescence was quenched by functional depletion of Zn using a membrane-permeable Zn chelator N, N, N′, N′-tetrakis (2-pyridyl-methyl)ethylenediamine (TPEN). Zn levels were also depleted by various mast cell activators, including IgE/anti-IgE, and Zn was rapidly replenished during subsequent culture, suggesting an active uptake mechanism. In support of the latter, mast cells contained high levels of the vesicular Zn transporter ZnT4, especially in the more apical granules. Immunofluorescence and immunogold labeling studies revealed significant pools of procaspase-3 and -4 in mast cell granules and their release during degranulation. Functional depletion of Zn by chelation with TPEN, but not by degranulation, resulted in greatly increased susceptibility of mast cells to toxin-induced caspase activation, as detected using a fluorogenic substrate assay. Release of caspases during degranulation was accompanied by a decreased susceptibility to toxins. Zn depletion by chelation, but not by degranulation, also resulted in nuclear translocation of the antiapoptotic, proinflammatory transcription factor NF-κB. These findings implicate a role for ZnT4 in mast cell Zn homeostasis and suggest that granule pools of Zn may be distinct from those regulating activation of procaspase-3 and NF-κB.

Use of a zinc fluorophore to measure labile pools of zinc in body fluids and cell-conditioned media

Here we describe a rapid and sensitive zinquin-based fluorometric assay that enables one to monitor levels of labile Zn(II) in body fluids, buffers, and cell-conditioned culture media as well as changes in these pools in disease. Labile pools of Zn(II) are free or loosely bound pools and more tightly bound but zinquin-accessible pools in contrast to the fixed pools of Zn(II) within metalloproteins. In human plasma, mean labile Zn(II) was 8.1 microM (SEM 0.53; n = 81) and constituted about 70% of the total plasma Zn(II) and >90% of human plasma albumin Zn(II). Plasma labile Zn(II) was significantly depleted after 7 days of Zn(II) deprivation in mice, despite only small changes in body weight. Labile Zn(II) concentrations were also measured in the induced sputum plugs, saliva, and urine of normal adults and were 1.30 microM (SEM 0.27; n = 73), 0.11 microM (SEM 0.11; n = 6), and 0.23 microM (SEM 0.08; n = 8), respectively. Urinary labile Zn(II) concentration was significantly increased in some patients with type II diabetes mellitus (overall mean was 0.90 microM, SEM 0.30; n = 12). The technique may be particularly useful in assessing extracellular Zn(II) levels in diseases associated with altered Zn(II) homeostasis, identifying those subjects most in need of Zn(II) supplementation, and defining the optimum concentrations of available Zn(II) in buffers and culture media.

Zinc and its Specific Transporters as Potential Targets in Airway Disease

The dietary group IIb metal zinc (Zn) plays essential housekeeping roles in cellular metabolism and gene expression. It regulates a number of cellular processes including mitosis, apoptosis, secretion and signal transduction as well as critical events in physiological processes as diverse as insulin release, T cell cytokine production, wound healing, vision and neurotransmission. Critical to these processes are the mechanisms that regulate Zn homeostasis in cells and tissues. The proteins that control Zn uptake and compartmentalization are rapidly being identified and characterized. Recently, the first images of sub-cellular pools of Zn in airway epithelium have been obtained. This review discusses what we currently know about Zn in the airways, both in the normal and inflamed states, and then considers how we might target Zn metabolism by developing strategies to monitor and manipulate airway Zn levels in airway disease.

Zinc and Zinc Transporters in Macrophages and Their Roles in Efferocytosis in COPD

Our previous studies have shown that nutritional zinc restriction exacerbates airway inflammation accompanied by an increase in caspase-3 activation and an accumulation of apoptotic epithelial cells in the bronchioles of the mice. Normally, apoptotic cells are rapidly cleared by macrophage efferocytosis, limiting any secondary necrosis and inflammation. We therefore hypothesized that zinc deficiency is not only pro-apoptotic but also impairs macrophage efferocytosis. Impaired efferocytic clearance of apoptotic epithelial cells by alveolar macrophages occurs in chronic obstructive pulmonary disease (COPD), cigarette-smoking and other lung inflammatory diseases. We now show that zinc is a factor in impaired macrophage efferocytosis in COPD. Concentrations of zinc were significantly reduced in the supernatant of bronchoalveolar lavage fluid of patients with COPD who were current smokers, compared to healthy controls, smokers or COPD patients not actively smoking. Lavage zinc was positively correlated with AM efferocytosis and there was decreased efferocytosis in macrophages depleted of Zn in vitro by treatment with the membrane-permeable zinc chelator TPEN. Organ and cell Zn homeostasis are mediated by two families of membrane ZIP and ZnT proteins. Macrophages of mice null for ZIP1 had significantly lower intracellular zinc and efferocytosis capability, suggesting ZIP1 may play an important role. We investigated further using the human THP-1 derived macrophage cell line, with and without zinc chelation by TPEN to mimic zinc deficiency. There was no change in ZIP1 mRNA levels by TPEN but a significant 3-fold increase in expression of another influx transporter ZIP2, consistent with a role for ZIP2 in maintaining macrophage Zn levels. Both ZIP1 and ZIP2 proteins were localized to the plasma membrane and cytoplasm in normal human lung alveolar macrophages. We propose that zinc homeostasis in macrophages involves the coordinated action of ZIP1 and ZIP2 transporters responding differently to zinc deficiency signals and that these play important roles in macrophage efferocytosis.

Intracellular zinc stores protect the intestinal epithelium from Ochratoxin A toxicity.

Ochratoxin A (OTA) is a harmful mycotoxin frequently contaminating foods, feeds and beverages. OTA was reported to be nephrotoxic, immunotoxic, hepatotoxic and a potential carcinogen, with yet poorly characterized mechanisms. Although intestinal cells are relatively resistant to high concentrations of OTA, interaction with other dietary factors or specific nutritional conditions may increase OTA toxicity to the intestinal mucosa. The role of intracellular zinc stores in protecting the integrity of intestinal mucosa has been investigated in human Caco-2/TC7 cells challenged with OTA. Zinc depletion of cells incubated with TPEN, a specific zinc chelator, caused an increase of tight junction permeability in OTA treated cells, accompanied by increased apoptosis. These effects were fully reverted by zinc supplementation during TPEN treatment, showing a specific role for this micronutrient in enterocyte defence mechanisms from OTA toxicity. A complex perturbation of zinc homeostasis was also demonstrated by analyzing the expression of genes coding for proteins involved in cellular zinc. In particular, zinc-dependent up-regulation of the metallothionein gene MT2A upon OTA treatment may indicate that the mycotoxin acts through generation of redox imbalance and that zinc deprivation reduces the intracellular defence mechanisms against noxious insults.

Transcriptional regulation of the ezrin gene during rat intestinal development and epithelial differentiation

Polarized intestinal epithelial cells are characterized by the presence of a brush border at their apical surface. The brush border cytoskeleton is assembled during cell differentiation and is composed of parallel actin bundles, held together by specific actin-binding proteins. Using specific cDNA probes we have studied the expression of the mRNAs encoding ezrin and moesin, two members of a class of proteins that connect the microvillar cytoskeleton to the plasma membrane, during the process of enterocyte maturation that occurs both in the embryonic and in the adult small intestine, along the crypt-villus axis. The steady state levels of ezrin mRNA were found to increase in the fetal gut epithelium between day 15 and day 20 of gestation and during the first week after birth, in parallel with the morphogenetic process that leads to cell polarization and brush border assembly. On the contrary, moesin mRNA is expressed at very low levels in the mature small intestine, with a sudden drop in transcription occurring at birth. In the continuously renewing epithelium of adult animals, ezrin mRNA levels are higher in the differentiated villus cells of the distal portions of the gastrointestinal tract and very low in undifferentiated crypt cells. These data demonstrate that the expression of the ezrin gene is regulated at the level of mRNA abundance during development and differentiation of the intestinal epithelium.

The Znt4 mutation inlethal milk mice affects intestinal zinc homeostasis through the expression of other Zn transporters.

The lethal milk mouse syndrome is caused by a point mutation in the zinc transporter gene ZnT4 resulting in defective zinc secretion in the milk of homozygous mutant dams. Pups of any genotype fed solely on lm milk die within the first two weeks of neonatal life, displaying zinc deficiency symptoms. Homozygous mutant pups survive when foster nursed by wild type dams and show signs of mild zinc deficiency in adulthood. To further investigate the role of ZnT4 in zinc secretion in the intestinal epithelium, we have studied the expression by real time quantitative PCR of mutant ZnT4 and of other zinc transporters of the Zip and ZnT families, in the jejunum of homozygous lm mice and of the isogenic wild type strain C57BL/ 6J. We report in this paper that expression of the mutant ZnT4 mRNA, carrying a premature translational termination codon (ZnT4/lm), is almost absent in tissues from lm mice, probably as a result of degradation by the Nonsense Mediated mRNA Decay (NMD) Pathway. In the jejunum of mutant mice, we also observed decreased expression of the uptake zinc transporter Zip4, paralleled by increased levels of both metallothionein genes MTI and MTII. Zinc supplementation of lm mice in the drinking water did not result in further decrease of Zip4 expression, but led to full induction of MT mRNAs. These results lead us to conclude that, although in the enterocytes of lm mice the absence of the zinc secretion activity mediated by ZnT4 results in increased intracellular zinc concentration, other zinc efflux activities are able to maintain the level of zinc ions below the threshold necessary for full induction of metallothioneins.

Consensus statement understanding health and malnutrition through a systems approach: the ENOUGH program for early life

Abstract Nutrition research, like most biomedical disciplines, adopted and often uses experimental approaches based on Beadle and Tatum’s one gene—one polypeptide hypothesis, thereby reducing biological processes to single reactions or pathways. Systems thinking is needed to understand the complexity of health and disease processes requiring measurements of physiological processes, as well as environmental and social factors, which may alter the expression of genetic information. Analysis of physiological processes with omics technologies to assess systems’ responses has only become available over the past decade and remains costly. Studies of environmental and social conditions known to alter health are often not connected to biomedical research. While these facts are widely accepted, developing and conducting comprehensive research programs for health are often beyond financial and human resources of single research groups. We propose a new research program on essential nutrients for optimal underpinning of growth and health (ENOUGH) that will use systems approaches with more comprehensive measurements and biostatistical analysis of the many biological and environmental factors that influence undernutrition. Creating a knowledge base for nutrition and health is a necessary first step toward developing solutions targeted to different populations in diverse social and physical environments for the two billion undernourished people in developed and developing economies.

Ultrastructural analysis, zinc transporters, glucose transporters and hormones expression in New world primate (Callithrix jacchus) and human pancreatic islets.

The New world primates (NWP) Callithrix jacchus separated from man approximately 50 million years ago and is a potential alternative small non-human primate model for diabetes research. Ultrastructure, and gene expression of pancreatic islets and the recently described diabetes auto antigenic zinc transporters families in human, NWP and pig pancreas were studied. Morphologically NWP islets were larger than pig islets and similar in size to human islets. NWP islets alpha cells had high dense core surrounded by a limiting membrane, beta cells by the mixed morphology of the granule core, and delta cells by moderate opaque core. Antibody staining for insulin, glucagon, somatostatin and Glucagon-like peptide-1 (GLP-1) showed that the distribution pattern of the different cell types within islets was comparable to pig and human islets. In all three species protein expression of zinc transporter ZnT8 was detected in most of the insulin producing beta cells whereas Zip14 expression was widely expressed in alpha and beta cells. In both human and NWP little or no expression of Glut2 was observed compared to Glut1 and glucokinase at the protein level, however the messenger RNA level of Glut2 was greater than Glut1 and glucokinase. In contrast all three glucose transporters were expressed in pig islets at the protein level. The expression of Zip14 in islets is reported for the first time. In conclusion NWP pancreatic islets express comparable islet cell types and distribution to humans and pigs. Importantly, marmosets have a similar glucose transporter profile to humans, making this non-endangered primate species a useful animal model for pancreatic biology.