Soybean extracts increase cell surface ZIP4 abundance and cellular zinc levels: a potential novel strategy to enhance zinc absorption by ZIP4 targeting.

Dietary zinc deficiency puts human health at risk, so we explored strategies for enhancing zinc absorption. In the small intestine, the zinc transporter ZIP4 functions as an essential component of zinc absorption. Overexpression of ZIP4 protein increases zinc uptake and thereby cellular zinc levels, suggesting that food components with the ability to increase ZIP4 could potentially enhance zinc absorption via the intestine. In the present study, we used mouse Hepa cells, which regulate mouse Zip4 (mZip4) in a manner indistinguishable from that in intestinal enterocytes, to screen for suitable food components that can increase the abundance of ZIP4. Using this ZIP4-targeting strategy, two such soybean extracts were identified that were specifically able to decrease mZip4 endocytosis in response to zinc. These soybean extracts also effectively increased the abundance of apically localized mZip4 in transfected polarized Caco2 and Madin-Darby canine kidney cells and, moreover, two apically localized mZip4 acrodermatitis enteropathica mutants. Soybean components were purified from one extract and soyasaponin Bb was identified as an active component that increased both mZip4 protein abundance and zinc levels in Hepa cells. Finally, we confirmed that soyasaponin Bb is capable of enhancing cell surface endogenous human ZIP4 in human cells. Our results suggest that ZIP4 targeting may represent a new strategy to improve zinc absorption in humans.

Biliary excretion of essential trace elements in rats under oxidative stress caused by selenium deficiency.

The excretion of essential trace elements, namely, Se, Sr, As, Mn, Co, V, Fe, and Zn into the bile of Se-deficient (SeD) Wistar male rats was studied using the multitracer (MT) technique, and instrumental neutron activation analysis (INAA). Normal and Se-control (SeC) rat groups were used as reference groups to compare the effects of Se levels on the behaviors of the essential trace elements. The excretion (% dose) of Se, Sr, As, Mn, Co, and V increased with Se levels in the liver. The biliary excretion of Mn and As dramatically enhanced for SeC rats compared with SeD rats, while that of V accelerated a little for SeC rats. The radioactivity levels of (59)Fe and (65)Zn in the MT tracer solution were insufficient to measure their excretion into bile. The role of glutathione and bilirubin for biliary excretion of the metals was discussed in relation to Se levels in rat liver.

Tissue nonspecific alkaline phosphatase is activated via a two-step mechanism by zinc transport complexes in the early secretory pathway.

A number of enzymes become functional by binding to zinc during their journey through the early secretory pathway. The zinc transporters (ZnTs) located there play important roles in this step. We have previously shown that two zinc transport complexes, ZnT5/ZnT6 heterodimers and ZnT7 homo-oligomers, are required for the activation of alkaline phosphatases, by converting them from the apo- to the holo-form. Here, we investigated the molecular mechanisms of this activation. ZnT1 and ZnT4 expressed in chicken DT40 cells did not contribute to the activation of tissue nonspecific alkaline phosphatase (TNAP). The reduced activity of TNAP in DT40 cells deficient in both ZnT complexes was not restored by zinc supplementation nor by exogenous expression of other ZnTs that increase the zinc content in the secretory pathway. Moreover, we showed that expression of ZnT5/ZnT6 heterodimers reconstituted with zinc transport-incompetent ZnT5 mutant failed to restore TNAP activity but could stabilize the TNAP protein as the apo-form, regardless of zinc status. These findings demonstrate that TNAP is activated not simply by passive zinc binding but by an elaborate two-step mechanism via protein stabilization followed by enzyme conversion from the apo- to the holo-form with zinc loaded by ZnT complexes in the early secretory pathway.

In vivo multitracer analysis technique: screening of radioactive probes for noninvasive measurement of physiological functions in experimental animals.

A novel screening experiment, to find radioactive probes for non-invasive measurements of physiological functions in experimental animals, was tested using the in vivo multitracer analysis technique. The details of the efficiency of the detector settings used in the in vivo multitracer analysis technique were examined by both computer simulations and practical measurements. Multiple radioactive isotopes, i.e. multitracer, were prepared by irradiating a silver foil target with a heavy ion beam at the RIKEN ring cyclotron. After chemical separation of the silver target, the multitracer was finally dissolved in isotonic citrate buffer. The multitracer solution was intravenously injected into rats. Using a gamma-ray detector equipped with a well-defined slit, the collimated gamma-rays from the upper abdomen of living rats were measured. After correction of detection efficiencies, it was possible to compare the distribution of radioactive elements between two groups of rats different in body weight. The in vivo measurement showed that the tissue substantial volume of the selenium-deficient (SeD) rat liver increased compared to normal rats. The possibility of a functional estimation of tissue/blood volume for living rats was proposed based on the characteristic in vivo distribution of 74As, 83Rb and 103Ru.

Distribution of trace elements in the brain of EL (epilepsy) mice.

The association of essential trace elements with epileptic seizures is poorly understood. On the basis of the evidences that the release of zinc from the brain of epilepsy (EL) mice, an animal model of genetically determined epilepsy, is enhanced by the induction of seizures and that alteration of zinc homeostasis is responsive to susceptibility to seizures, the distribution of trace elements in the brain was studied using EL mice and ddY mice, which form the genetic background for the inbred EL mice. The multitracer technique was applied to determine the distribution of trace elements. Twenty-four hours after intravenous injection of the multitracer, the concentration of 65Zn and 56Co in the brain of untreated EL mice was higher than in ddY mice, while the concentration of 65Zn and 56Co in the brain was decreased in seized EL mice. 75Se concentration in the hippocampus, cerebral cortex and cerebellum of untreated EL mice was lower than in ddY mice, while 75Se concentration in the hippocampus was increased in seized EL mice. 83Rb, an element of homologous series to potassium, concentration in the hippocampus and cerebral cortex of untreated EL mice was lower than in ddY mice, and 83Rb concentration in the cerebral cortex was decreased in seized EL mice. The movement of zinc, cobalt and selenium in the brain may be altered by enhancement of susceptibility to seizures. These results suggest that alteration of homeostasis of zinc, cobalt and selenium in the brain may be involved in the susceptibility, development or termination of seizures in EL mice.

[Noninvasive detection of dynamics of various elements in the upper abdomen of selenium-deficient rat using multitracer analysis technique].

The advantages of a technique as a diagnostic tool for examining the distribution of bio-trace elements in the living body are discussed. Time courses of the distribution of Be, V, Mn, Fe, Co, Zn, As, Se, Rb, Sr, and Y in the upper abdomen of living selenium-deficient rats were examined using the in vivo multitracer analysis technique. The dynamics of the elements were estimated by comparison with the distribution of As. Almost all As was taken up by red blood cells. The present findings of a decrease in Se and increase in Co in the liver of Se-deficient rats are in good agreement with the previous data that showed a decrease in Se and increase in Co uptake into the liver cell fraction of Se-deficient rats. Although the normalized uptake rate and the relative distribution of Co 48 h after administration increased in Se-deficient rats, the early level of the relative distribution of Co was not lower compared with that in the normal group. This suggests that the high level of the normalized uptake rate and the relative distribution of Co in Se-deficient rats were affected by the decreasing excretion rate rather than by the increasing uptake rate of Co. The plateau level of relative distribution of Se in the Se-deficient rats is lower than that in normal rats, suggesting that the lower levels of the normalized uptake rate and relative distribution of Se in Se-deficient rats were due to the decreased uptake rate of the element.