The Zinc Transporter Zip5 (Slc39a5) Regulates Intestinal Zinc Excretion and Protects the Pancreas against Zinc Toxicity

Background

ZIP5 localizes to the baso-lateral membranes of intestinal enterocytes and pancreatic acinar cells and is internalized and degraded coordinately in these cell-types during periods of dietary zinc deficiency. These cell-types are thought to control zinc excretion from the body. The baso-lateral localization and zinc-regulation of ZIP5 in these cells are unique among the 14 members of the Slc39a family and suggest that ZIP5 plays a role in zinc excretion.

Methods/Principal Findings

We created mice with floxed Zip5 genes and deleted this gene in the entire mouse or specifically in enterocytes or acinar cells and then examined the effects on zinc homeostasis. We found that ZIP5 is not essential for growth and viability but total knockout of ZIP5 led to increased zinc in the liver in mice fed a zinc-adequate (ZnA) diet but impaired accumulation of pancreatic zinc in mice fed a zinc-excess (ZnE) diet. Loss-of-function of enterocyte ZIP5, in contrast, led to increased pancreatic zinc in mice fed a ZnA diet and increased abundance of intestinal Zip4 mRNA. Finally, loss-of-function of acinar cell ZIP5 modestly reduced pancreatic zinc in mice fed a ZnA diet but did not impair zinc uptake as measured by the rapid accumulation of ⁶⁷zinc. Retention of pancreatic ⁶⁷zinc was impaired in these mice but the absence of pancreatic ZIP5 sensitized them to zinc-induced pancreatitis and exacerbated the formation of large cytoplasmic vacuoles containing secretory protein in acinar cells.

Conclusions

These studies demonstrate that ZIP5 participates in the control of zinc excretion in mice. Specifically, they reveal a paramount function of intestinal ZIP5 in zinc excretion but suggest a role for pancreatic ZIP5 in zinc accumulation/retention in acinar cells. ZIP5 functions in acinar cells to protect against zinc-induced acute pancreatitis and attenuate the process of zymophagy. This suggests that it may play a role in autophagy.     

The role of zinc in the iliac absorption of various L-tryptophane forms in chickens]

Zinc was found to enhance absorption of free and "peptide" L-tryptophan across the chick mucosal brush border. Intestinal absorption of free L-tryptophan can be partially inhibited by excessive amount of zinc. The findings suggest that zinc interacts with free and "peptide" L-tryptophan transports protecting them against degradation.     

Disorders of zinc transport through the ileal epithelium in vitamin A deficiency and ischemia

By means of histochemical revealing of zinc and applying radioactive isotope 65Zn accumulation, absorption and distribution of cations of this metal in the mucous membrane of the iliac intestine have been studied in chicken, normal, at ischemia and at A-avitaminosis. An essential zinc-depositing ability is peculiar to coverings of mucus upon the intestine epithelium, and among intracellular components--to smooth endoplasmic reticulum. A-avitaminosis and especially ischemia result in increasing permeability of the apical part of the external membrane of epitheliocytes, in overloading of the latter with zinc cations, when they are introduced into the intestine, as well as in decreasing transepithelial transport of zinc. The changes mentioned are accompanied with certain disturbances in the barrier function of the intestinal epithelium.     

Role of exchange transport in the absorption of L-tryptophan in the small intestine of chicks

1. In in vitro experiments with accumulating mucosal preparations (AMP) and everted intestinal sacs, as well as in in vivo experiments with isolated loops of the small intestine the stimulating effect of a number of amino acids on L-tryptophan uptake was investigated. 2. Under "switched off" active transport (anoxia, 2,4-DNF treatment, sodium ion replacement by lithium ions in the mucosal solution) an expressed stimulation of L-tryptophan transport was observed within the mucosa and across the wall of the small intestine in the presence of L-proline, glycine, L-alpha-alanine, L-histidine and L-lysine. 3. Preincubation of AMP in the solutions of glycine, L-alpha-alanine and L-lysine was characterized by a stimulation of L-tryptophan transport, and the increase of its concentration in tissue was accompanied by the exit of an equivalent amount of glycine from it. 4. These observations show the participation of exchange transport in the uptake of L-tryptophan in the small intestine of chicks. 5. The mechanism of exchange transport in chicks starts to function on the 25th day after hatching and its intensity depends on the character of amino acid-modifier participating in the process. 6. Maximum activity of the exchange transport of L-tryptophan is demonstrated in the middle ileum. 7. L-alpha-Alanine stimulates the absorption of L-tryptophan from the isolated intestinal loop proving the existence of an exchange transport mechanism in a living organism. 8. An increased intensity of exchange transport is observed when feeding chicks with diets deficient and enriched in tryptophan.     

The bile acid receptor GPBAR-1 (TGR5) modulates integrity of intestinal barrier and immune response to experimental colitis

Background

GP-BAR1, a member G protein coupled receptor superfamily, is a cell surface bile acid-activated receptor highly expressed in the ileum and colon. In monocytes, ligation of GP-BAR1 by secondary bile acids results in a cAMP-dependent attenuation of cytokine generation.

Aims

To investigate the role GP-BAR1 in regulating intestinal homeostasis and inflammation-driven immune dysfunction in rodent models of colitis.

Methods

Colitis was induced in wild type and GP-BAR1^−/− mice by DSS and TNBS administration. Potential GP-BAR1 agonists were identified by in silico screening and computational docking studies.

Results

GP-BAR1^−/− mice develop an abnormal morphology of colonic mucous cells and an altered molecular architecture of epithelial tight junctions with increased expression and abnormal subcellular distribution of zonulin 1 resulting in increased intestinal permeability and susceptibility to develop severe colitis in response to DSS at early stage of life. By in silico screening and docking studies we identified ciprofloxacin as a GP-BAR1 ligand. In monocytes, ciprofloxacin increases cAMP concentrations and attenuates TNFα release induced by TLR4 ligation in a GP-BAR1 dependent manner. Treating mice rendered colitic by TNBS with ciprofloxacin and oleanolic acid, a well characterized GP-BAR1 ligand, abrogates signs and symptoms of colitis. Colonic expression of GP-BAR1 mRNA increases in rodent models of colitis and tissues from Crohn''s disease patients. Flow cytometry analysis demonstrates that ≈90% of CD14+ cells isolated from the lamina propria of TNBS-treated mice stained positively for GP-BAR1.

Conclusions

GP-BAR1 regulates intestinal barrier structure. Its expression increases in rodent models of colitis and Crohn''s disease. Ciprofloxacin is a GP-BAR1 ligand.     

Coupling of protease activity and sodium loading with intestinal absorption of amino acids

Membrane-bound serine proteases to play a certain role in activation of sodium transport in epithelial cells. To were found explain the protease activity and sodium-dependent L-tryptophan transport across chicken small intestine interaction, four experiments were conducted. One hundred chicks were fed diets that contained 0; 0.3; 3 or 6% of supplemental NaCl and were given distillated water ad libitum. Signs of salt toxicity observed were as follows: a decreased body weight, increased heart and kidney weights, formation of secondary lysosomes in enterocytes and lymphocytes. Such chickens were in the state of negative nitrogen balance. Intestinal absorption of L-tryptophan correlated with mucosal protease activity during increased dietary sodium chloride intake. Recent in vitro and in vivo experiments indicate that enterocyte proteases may be of critical importance in activation of sodium-dependent intestinal transporters for L-tryptophan.     

Intestinal zinc uptake in two marine teleosts,squirrelfish (Holocentrus adscensionis) and gulf toadfish (Opsanus beta)

Zinc is a vital micronutrient, yet as an environmental toxicant it can be deleterious to aquatic organisms such as fish. Consequently, the study of zinc uptake mechanisms is essential for understanding nutrition, toxicity, and metabolism of this metal. Intestinal zinc uptake was studied in two marine teleosts, using both in vitro (in vitro perfusion and intestinal sacs) and in vivo techniques (in situ bolus). Female squirrelfish (Holocentrus adscensionis) exhibited significantly increased epithelial zinc uptake associated with enhanced hepatic zinc accumulation. This confirms this zinc-hyperaccumulating teleost as a potential model of zinc absorption. Intestinal zinc uptake in the gulf toadfish (Opsanus beta) was biphasic with respect to zinc concentration (0.3-500 microM), exhibiting both saturable and passive uptake components. In both species, the passage of zinc into the postintestinal compartment was highly dependent on technique. Decreased proportions of postintestinal zinc in vivo, coupled with concentration-dependent distribution of zinc accumulation, suggested mechanisms may act to control the movement of zinc into the circulation. In addition, the results of this study were used to reinterpret previous findings of zinc uptake in freshwater fish and allowed a critique of techniques used to study intestinal metal uptake.     

Effects of vitamin A-deprivation on hamster tracheal epithelium. A quantitative morphologic study

The effects of vitamin A-deprivation on the tracheal epithelium were studied in 35-day old hamsters that had been raised since birth on a vitamin A-deficient diet. Colchicine and 3HTdR were given 6 hours before death and the proliferative activities of basal cells and mucous cells were quantified separately by 3HTdR labeling indices and mitotic rates. Vitamin A-deprivation decreased replication of basal cells and mucous cells in tracheal epithelium which showed minimal morphologic change. The mitotic rates and labeling indices were reduced 3 to 4-fold in basal cells and 14-fold in mucous cells (analyzed as percent of total number of each cell type) compared with controls. Thus, replication of mucous cells was more inhibited by lack of vitamin A, than replication of basal cells. The disparate hypoplasia of basal cells and mucous cells in epithelium showing minimal change, resulted in a relative increase in the proportion of basal cells and a relative decrease in the proportion of mucous cells, which could be erroneously interpreted as "basal cell hyperplasia". Proportions of preciliated and ciliated cells were also decreased compared to controls. At foci of stratification and epidermoid metaplasia, cell replication rates were increased over controls and more than 70% of all mitotic activity was associated with "non-basal" cells. Genesis of these lesions was coincident with cell death and cell loss. The histogenesis of stratification and epidermoid metaplasia was characterized. Morphological evidence indicated that these lesions were closely related histogenetically and were composed, for the most part, of altered mucous cells which expressed dual phenotypes i.e. keratinization and mucus synthesis.     

Effects of arachidonic acid and cyclo (his-pro) on zinc transport across small intestine and muscle tissues

Previously we have shown that arachidonic acid (AA) plus zinc or cyclo (his-pro) (CHP) plus zinc improve clinical signs of diabetes in streptozotocin-induced diabetic rats. Since streptozotocin destroys pancreatic beta-cells, we hypothesize that the effect of either AA or CHP, plus zinc on glucose metabolism is via mobilization of intracellular zinc which in turn stimulates glucose uptake by peripheral tissues. We now report the relationship between zinc and AA and between zinc and CHP in controlling zinc influx and efflux across hindlimb muscle cells isolated from three-month old rats. Although CHP increased muscle zinc influx in a dose-dependent manner, AA was not effective. However, AA was more effective in stimulating zinc efflux than CHP. We have previously demonstrated that AA stimulates intestinal zinc uptake and absorption, and now present evidence that CHP also influences intestinal zinc transport. These results suggest that both AA and CHP affect glucose uptake in muscle cells via stimulating intestinal zinc absorption and muscle cell zinc flux.     

The protective roles of NLRP6 in intestinal epithelial cells

The evolution of chronic inflammatory diseases is thought to be due to a combination of host genetic variations and environmental factors that include the alteration of intestinal flora, termed “dysbiosis.” The intestinal mucosal barrier includes a chemical barrier and physical barrier that have important roles in protecting the intestine against inflammatory injury. The chemical barrier includes antimicrobial peptides (AMPs), and the physical barrier includes a mucous layer, a monolayer of intestinal epithelial cells and cell junctions. The intestinal mucosal barrier is not a static barrier, but rather, it strongly interacts with the gut microbiome and cells of the immune system. Correct expression of AMPs, together with mucus and balanced epithelial cell proliferation, prevents the occurrence of disease. NLRP6, a member of the nucleotide‐binding domain, leucine‐rich repeat‐containing (NLR) innate immune receptor family, participates in the progression of intestinal inflammation and enteric pathogen infections. It has become apparent in recent years that NLRP6 is important in disease pathogenesis, as it responds to internal ligands that lead to the release of AMPs and mucus, thus regulating the regeneration of intestinal epithelial cells. This review summarizes the activation of NLRP6 and its protective role in the intestinal epithelial cell.     

Absorption and localization of zinc in the ileum of chicks dependent on provision with vitamin A

Vitamin A increase the accumulation and transport of zinc in the chick ileal mucosa. Regulating effect of this vitamin on the mentioned processes was already observed in the initial stage zinc uptake in the intestinal mucosa--its storage in the mucus layer. Stimulation of zinc absorption by vitamin A can be related to a specific carrier-vitamin A-dependent zinc-binding protein. Presence of zinc in the smooth endoplasmic reticulum of the enterocyte apical zone suggests the role of this organelle in the cation homeostasis in the cell.     

Specialized mucous glands and their possible adaptive role in the males of some species of Rana (Amphibia,Anura)

A structural and ultrastructural study was carried out on the cutaneous glands in some species of Rana (R. dalmatina, R. iberica, R. italica, R. "esculenta," and R. perezi), giving particular attention to the mucous secretory units. Two different types of mucous glands occur in R. dalmatina, R. iberica, and R. italica. Besides the ordinary mucous units, which are randomly distributed over the body surface in both males and females, a further population of mucous glands was observed on the male dorsal skin. The latter is recognizable by the peculiar morphology of the epithelial cells and some characteristics of the secretory product. Specialized mucous glands are absent in both sexes of R. "esculenta" and R. perezi. The possible adaptive role of the specialized mucous glands is discussed in light of the absence of vocal sacs in males of R. dalmatina, R. iberica, and R. italica. Chemosignals released by sexually dimorphic mucous units may replace vocal communication during the breeding season and so play an important role in female attraction and/or territorial announcement. The morphology and possible function of the specialized mucous glands in the three species of Rana are compared with the breeding glands of other frogs and with the hedonic glands of some urodeles (Salamandridae and Plethodontidae), which are known to produce pheromonal substances during courtship.     

New ways of thinking about (and teaching about) intestinal epithelial function

This article summarizes a presentation made at the Teaching Refresher Course of the American Physiological Society, which was held at the Experimental Biology meeting in 2007. The intestinal epithelium has important ion transport and barrier functions that contribute pivotally to normal physiological functioning of the intestine and other body systems. These functions are also frequently the target of dysfunction that, in turn, results in specific digestive disease states, such as diarrheal illnesses. Three emerging concepts are discussed with respect to ion transport: the complex interplay of intracellular signals that both activate and inhibit chloride secretion; the role of multiprotein complexes in the regulation of ion transport, taking sodium/hydrogen exchange as an example; and acute and chronic regulation of colonic sodium absorption, involving both sodium channel internalization and de novo synthesis of new channels. Similarly, recently obtained information about the molecular components of epithelial tight junctions and the ways in which tight junctions are regulated both in health and disease are discussed to exemplify ways to teach about intestinal barrier properties. Finally, both genetically determined intestinal diseases and those arising as a result of infections and/or inflammation are described, and these can be used as the means to enhance the basic and clinical relevance of teaching about intestinal epithelial physiology as well as the impact that the understanding of such physiology has had on associated therapeutics. The article also indicates, where relevant, how different approaches may be used effectively to teach related concepts to graduate versus medical/professional student audiences.     

Effect of nicotinic acid on zinc and iron metabolism

Nicotinic acid has functional groups capable of forming complexes with trace metals. The present study examines the effect of nicotinic acid supplementation on absorption and utilization of zinc and iron. In vitro zinc uptake by human erythrocytes was studied using blood samples of 10 healthy subjects. It was found that 8 moles nicotinic acid or NADP increased ⁶⁵Zn uptake by 38.9% and 43.1% in fasting, and by 70.9% and 28.1% in postprandial conditions. In animal experiments, nicotinic acid supplementation to finger millet based diet resulted in significant enhancement of percent zinc absorption, liver zinc and growth of weanling mice (P < 0.05). When mice were fed with nicotinic acid-deficient, -adequate and -excess synthetic diets for four weeks it was observed that, in comparison with the nicotinic acid-deficient diet, percent zinc absorption, intestinal zinc, percent haeomoglobin and liver iron increased significantly under nicotinic acid-adequate and -excess conditions. The results obtained suggested that nicotinic acid, in addition to its known effect on growth and metabolism, may be playing an important role in enhancing zinc and iron utilization.     

White monkey syndrome in infant baboons (Papio species)

Over 23 months, zinc toxicosis was diagnosed in 35 baboons aged 5-12 months in one galvanized metal and concrete cage complex with conditions that led to excessive exposure to environmental zinc. Clinical signs included reduced pigmentation of hair, skin, and mucous membranes (whiteness), alopecia, dehydration, emaciation, cachexia, dermatitis, diarrhea and, in six cases, severe gangrenous dermatitis of extremities. The syndrome was characterized by pancytopenia, elevated zinc and low copper serum concentrations, low vitamin D and bone-specific alkaline phosphatase levels, and atypical myelomonocytic proliferation of bone marrow. This syndrome emphasizes the importance of proper husbandry and cage design and indicates the potential of infant baboons as a model to study the effects of excessive zinc on development. This is the first report describing the epidemiologic and clinical presentation of zinc toxicosis in infant baboons in captivity.     

Waterborne zinc alters temporal dynamics of guppy Poecilia reticulata epidermal response to Gyrodactylus turnbulli (Monogenea)

The present study assessed the histological changes in the epidermis of Poecilia reticulata induced by the combined effects of an ectoparasite Gyrodactylus turnbulli and differing concentrations of waterborne zinc (Zn). Infected guppies were exposed to 0, 15, 30, 60, or 120 μg Zn l-1 and monitored over 3 wk during the exponential increase in parasite numbers on the fish. The fish epidermis responded within 3 d to G. turnbulli infection with a rapid increase in epidermal thickness and a modest increase in number, but not size or composition, of mucous cells. In contrast, in the presence of combined waterborne Zn and infection, mucous cell numbers declined rapidly. As the parasite numbers increased, the epidermis remained thicker than normal, and the number and size of mucous cells decreased. The addition of Zn led to a dramatic thickening of the epidermis during the exponential growth of the parasite population. Mucous cell numbers remained depressed. Temporal changes in mucous cell size were Zn concentration dependent. At 60 μg Zn l-1, cells returned to normal size as infection progressed, whereas they remained extremely small at 120 μg Zn l-1. Changes in mucin composition previously reported in response to Zn alone were subdued in the presence of the parasite except at 60 μg Zn l-1, where all cells contained only acidic mucins. Together these results demonstrate that, on exposure to both Zn and G. turnbulli infection, the epidermal response is initially a protective response to both stressors, and then mainly driven by the increased parasite burden.     

异麦芽低聚糖对D-半乳糖致衰老大鼠肠道菌群、血清IgG和肠黏膜sIgA的影响

目的探讨异麦芽低聚糖对D-半乳糖致衰老大鼠肠黏膜功能的影响。方法Wistar大鼠随机分为3组：（1）青年对照组,（2）衰老对照组,（3）衰老观察组（衰老＋异麦芽低聚糖）。采用D-半乳糖造成衰老模型后,应用异麦芽低聚糖灌胃,检测各组肠道菌群、血清IgG和肠黏膜sIgA。结果D-半乳糖致衰老大鼠肠道菌群失调;灌胃异麦芽低聚糖后,衰老大鼠肠道双歧杆菌增加（P〈0．05）,肠杆菌和肠球菌数量减少（P〈0．05）;血清IgG、肠黏膜sIgA含量增加（P〈0．05）。结论异麦芽低聚糖可改善衰老机体肠黏膜功能。     

Regulation of adherens junctions and epithelial paracellular permeability: a novel function for polyamines

Maintenance of intestinal mucosal epithelial integrity requires polyamines that are involved in the multiple signaling pathways controlling gene expression and different epithelial cell functions. Integrity of the intestinal epithelial barrier depends on a complex of proteins composing different intercellular junctions, including tight junctions, adherens junctions, and desmosomes. E-cadherin is primarily found at the adherens junctions and plays a critical role in cell-cell adhesions that are fundamental to formation of the intestinal epithelial barrier. The current study determined whether polyamines regulate intestinal epithelial barrier function by altering E-cadherin expression. Depletion of cellular polyamines by alpha-difluoromethylornithine (DFMO) reduced intracellular free Ca2+ concentration ([Ca2+]cyt), decreased E-cadherin expression, and increased paracellular permeability in normal intestinal epithelial cells (IEC-6 line). Polyamine depletion did not alter expression of tight junction proteins such as zona occludens (ZO)-1, ZO-2, and junctional adhesion molecule (JAM)-1. Addition of exogenous polyamine spermidine reversed the effects of DFMO on [Ca2+]cyt and E-cadherin expression and restored paracellular permeability to near normal. Elevation of [Ca2+]cyt by the Ca2+ ionophore ionomycin increased E-cadherin expression in polyamine-deficient cells. In contrast, reduction of [Ca2+]cyt by polyamine depletion or removal of extracellular Ca2+ not only inhibited expression of E-cadherin mRNA but also decreased the half-life of E-cadherin protein. These results indicate that polyamines regulate intestinal epithelial paracellular barrier function by altering E-cadherin expression and that polyamines are essential for E-cadherin expression at least partially through [Ca2+]cyt.     

Infrared spectra of carbon monoxide complexes of indoleamine 2,3-dioxygenase and L-tryptophan 2,3-dioxygenases. Effects of substrates on the CO-stretching frequencies

Carbonmonoxy indoleamine 2,3-dioxygenase from rabbit small intestine exhibited two CO stretch bands at 1953 and 1933 cm-1 with half-band widths (delta v 1/2) of both approximately 15 cm-1. Upon addition of an excess amount of L-tryptophan, the substrate, the spectrum changed into that with an intense single band at 1902 cm-1 with the delta v 1/2 of 15 cm-1. Carbonmonoxy L-tryptophan 2,3-dioxygenase of Pseudomonas acidovorans in the absence of L-tryptophan showed a fused CO stretch band which consists of two components at 1965 and 1958 cm-1 (delta v 1/2 for the fused band; 25 cm-1), which was converted into a sharp single band at 1968 cm-1 (delta v 1/2; 10 cm-1) upon addition of excess L-tryptophan. On the other hand, CO complex of rat liver L-tryptophan 2,3-dioxygenase in the absence of L-tryptophan gave a spectrum with a poorly defined peak around 1961 cm-1. By the addition of L-tryptophan, the spectrum changed into that with two distinct bands at 1972 and 1920 cm-1 (delta v 1/2; 6 and 13 cm-1, respectively). These spectra were insensitive to pH in a range where the enzymes were not denatured (neutral to near pH 9). The infrared spectra of the carbonmonoxy enzymes were also affected by the addition of certain effectors such as skatole and alpha-methyl-DL-tryptophan, which facilitate the binding of L-tryptophan to the catalytic site of intestinal and Pseudomonas enzymes, respectively. However, the changes were of different types from those by the saturating amount of L-tryptophan. Possible mechanisms for these phenomena are discussed in relation to the structure of the heme-CO complex in these heme-containing dioxygenases.     

Ultrastructural study on route of gut bacterial translocation in a rat after spinal cord injury

Objective: To observe the ultrastructural change of the route of gut bacterial translocation in a rat with spinal cord injury(SCI).Methods: Forty Wistar rats were divided into the following groups: control group and 3 SCI groups(10 in each group). The rats in the SCI groups were established SCI model at 24 h, 48 h, and 72 h after SCI. Small intestine mucous membrane tissue was identified and assayed by transmission electron microscope, scanning electron microscope and immunofluorescence microscopy. Results: Small intestine mucous membrane tissue in control group was not damaged significantly, but those in SCI groups were damaged significantly. Proliferation bacteria in gut lumen attached on microvilli. The extracellular bacteria torn the intestinal barrier and perforated into the small intestinal mucosal epithelial cell. The bacteria and a lot of particles of the seriously damaged region penetrated into the lymphatic system and the blood system directly. Some bacteria were internalized into the goblet cell through the apical granule. Some bacteria and particles perforated into the submucosa of the M cell running the long axis of M cells through the tight junctions. In the microcirculation of mucosa, the bacteria that had already broken through the microvilli into blood circulation swim accompanying with erythrocytes. Conclusion: The routes of bacterial translocation interact and format a vicious circle. At early step, the transcellular pathway of bacterial translocation is major. Following with the destroyed small intestine mucous, the routes of bacterial translocation through the lymphatic system and the blood system become direct pathways. The goblet cell-dendritic cell and M cell pathway also play an important role in the bacterial translocation.