Affinity labeling of phosphoenolpyruvate carboxykinase with 1, 5-I-AEDANS.

The concentrations of zinc thionein and cytosolic zinc in rat liver were examined in male rats five days after bilateral adrenalectomy. Zinc in metallothionein increased 10 fold, as compared with control animals. Cytosolic zinc increased 79% as compared with controls. 65% of this increase could be accounted for bound to metallothionein. Sham operated animals after five days showed a 4 fold increase in hepatic zinc thionein and a 23% increase in cytosolic zinc, 71% of this increase being bound to metallothionein. Adrenalectomized rats, maintained on daily injections of corticosterone (4mg/100g b.w.), exhibited the same levels of zinc thionein and cytosolic zinc as adrenalectomized rats receiving no treatment. Adrenalectomized rats, maintained on daily injections of aldosterone (5μg/100g b.w.), exhibited the same levels of zinc thionein as the sham operated rats, but the cytosolic zinc remained elevated at the level found in adrenalectomized rats receiving no treatment. These results indicate that there is adrenal involvement in the control of hepatic zinc and zinc thionein levels in the rat.     

Regulation of liver metallothionein and plasma zinc by the glucocorticoid dexamethasone.

Administration of the glucocorticoid dexamethasone to adrenalectomized rats significantly decreased the serum zinc concentration within 14 hr. Dexamethasone did not detectably alter the liver zinc content, but markedly increased the proportion of zinc associated with liver metallothionein. The rate of incorporation of ³⁵S-cystine into this protein was stimulated to a maximal extent 7 hr after administration of the glucocorticoid. Poly(A)⁺ mRNA from liver polysomes was isolated and translated in a cell-free protein synthesizing system. Nearly twice as much polysomal metallothionein mRNA was found 7 hr following treatment with dexamethasone. These results suggest that glucocorticoids can regulate the plasma zinc concentration by a process that is related to the biosynthesis of the hepatic zinc-binding protein, metallothionein.     

Augmentation of dexamethasone induction of rat liver metallothionein by zinc.

The induction of liver metallothionein by dexamethasone in adrenalectomized rats was augmented by zinc administration. Metallothionein synthesis was increased in an additive manner with both zinc and dexamethasone compared with either treatment alone. Translational activity of polyribosomal metallothionein mRNA was also greater in zinc + dexamethasone-treated rats. Northern-blot analyses showed that dexamethasone increased these mRNA contents to a greater extent at the lower zinc dose, suggesting that the induction may be maximal at the higher zinc dose when combined with dexamethasone.     

Metallothionein mRNA levels are influenced by dietary cyclodextrins in rats

The relationship between metallothionein mRNA levels and the amounts of copper and zinc in liver, kidney and small intestine by feeding dietary cyclodextrin was examined in growing Wistar rats. alpha-, beta- or gamma-cyclodextrin was fed at 50 g/kg of diet for a 7-days period (ad libitum). After feeding, the liver zinc of rats fed beta-cyclodextrin was greater than those of rats fed the other three diets. Copper accumulated in kidney of rats fed alpha- or beta-cyclodextrin. Copper content in the small intestine did not show any alterations among rats fed all kinds of diets. The cyclodextrin-supplemented diets were ineffective in zinc content in every organ. There was the greatest level of copper in serum of rats fed beta-cyclodextrin, whereas the highest level of serum zinc was observed in rats fed gamma-cyclodextrin diet. Northern blot analysis demonstrated that dietary beta- and gamma-cyclodextrins, but not alpha-cyclodextrin markedly increased the metallothionein mRNA in the liver, whereas small intestinal metallothionein mRNA levels were markedly decreased. Kidney metallothionien mRNA levels were raised appreciably by all dietary cyclodextrin intakes. Metallothionein gene expressions in liver, kidney and small intestine were not proportional to liver and serum copper or zinc levels in those tissues. These results suggest that regulation of the metallothionein mRNA levels may at least partly involved with the accumulation of metals as copper in liver and kidney of rats fed cyclodextrins.     

Dietary beta- and gamma-cyclodextrins stimulation of hepatic metallothionein gene expression in rats

This study investigated whether hepatic metallothionein gene expression is affected by dietary cyclodextrins. Young male Wistar rats were fed a basal diet or cyclodextrin-supplemented (50 g of cyclodextrin per kg diet) diets for 7 d. Copper content in the liver did not show any significant changes among rats fed the basal, beta- and gamma-cyclodextrin diets. There were no differences in liver or serum zinc among groups. Copper content in serum was markedly decreased in rats fed the gamma-cyclodextrin-supplemented diet. Liver metallothionein mRNA levels were significantly elevated in both beta- and gamma-cyclodextrins-fed rats, but not in alpha-cyclodextrin-fed rats. Thus, the increase in hepatic metallothionein mRNA levels might be due to this mechanism except for the contents of copper and zinc in the liver.     

Apocynin Alleviates Renal Ischemia/Reperfusion Injury Through Regulating the Level of Zinc and Metallothionen

The purpose of this research was to evaluate the protective effects of apocynin on renal ischemia/reperfusion (I/R) injury (RI/RI) in rats. Rats preconditioned with apocynin were subjected to renal I/R. Zinc levels in serum and renal tissues, blood urea nitrogen (BUN), and serum creatinine (Scr) were detected. We further measured the activity of superoxide dismutase (SOD); the content of malondialdehyde (MDA), IL-4, IL-6, IL-10, and TNF-α; and the expression of metallothionein (MT) in the renal tissues. Results indicated that the levels of MDA, IL-4, IL-6, IL-10, TNF-α, and MT in the kidney tissue and serum BUN and Scr levels in RI/RI group were significantly higher than those in sham-operated group, while the levels of serum Zn and kidney Zn and SOD were reduced in RI/RI group. Apocynin treatment further decreased the levels of MDA, IL-6, TNF-α, and serum BUN and Scr, whereas it significantly increased the levels of Zn, SOD, IL-4, IL-10, and MT in the kidney tissue and serum Zn. These findings suggest that apocynin might play a protective role against RI/RI in rats through regulating zinc level and MT expression involving in oxidative stress.     

Differential effect of adrenalectomy on rat liver metallothionein mRNA levels in basal and stress conditions.

Liver metallothionein (MT) mRNA and serum MT levels of adrenalectomized (ADX) and sham-ADX rats in basal and stress (1, 3 or 6 h of restraint) conditions have been measured. Serum MT levels were overall lower in ADX than in sham-ADX rats. Basal liver MT mRNA levels were increased in ADX rats, suggesting that glucocorticoids have an inhibitory role on the regulation of liver MT synthesis. In contrast, liver MT mRNA levels were increased by stress in sham-ADX but not in ADX rats, suggesting a stimulatory role for glucocorticoids. These results suggest that glucocorticoids have a different role in liver MT regulation depending on the physiological situation.     

Metabolism of parenterally administered zinc and cadmium in livers of newborn rats

The interaction of injected zinc and cadmium with metallothionein was investigated in newborn rats. Tissues of 5-day-old rats were removed 24 h after a single injection (Sc) of saline or zinc (20 mg/kg, body wt.) or cadmium (1 mg/kg, body wt.) with 2.5 μCi of ⁶⁵Zn or ¹⁰⁹Cd or 5 μCi of [³⁵S]cysteine. Injection of zinc resulted in a 75% increase in the hepatic zinc concentration with a concomitant elevation of metallothionein (P < 0.001), zinc in metallothionein increased by 45% (P < 0.05); [³⁵S]cysteine incorporation indicated the induced synthesis of metallothionein. Injection of cadmium did not alter either metallothionein or zinc levels in liver, but cadmium in cytosol was preferentially bound to metallothionein. Neither treatment altered hepatic copper metabolism and copper in metallothionein, nor renal zinc and metallothionein levels. These data indicate that zinc injection can elevate hepatic zinc levels and induce metallothionein synthesis in newborn rats despite high basal levels; cadmium injection does not induce metallothionein synthesis, though cadmium is avidly sequestered by pre-existing metallothionein. The differences in the induction of metallothionein by these divalent cations can be explained by the differences in their binding affinities for thiol groups in intracellular metallothionein.     

Induction of metallothionein by exposure to normobaric 100% oxygen atmosphere in rats with different zinc supply

The objective of this study was to determine the effects of an oxygen enriched environment on the induction of the metalloprotein metallothionein (MT) and its relation to zinc metabolism in rats supplied with different levels of dietary zinc. Male albino rats were fed purified diets based on maize starch, egg white, saccharose and soybean oil differing in the concentration of zinc (1; 20; 100; 500 mg Zn/kg diet). At a dietary zinc supply of 1 mg/kg, the rats developed a zinc deficiency indicated by visual and biochemical parameters. At the end of the 37-day feeding period, half of the rats were exposed to 100% oxygen for 12 h.

The oxygen treatment significantly reduced plasma zinc in the zinc supplemented rats and reduced it in tendency in the zinc deficient rats. The MT concentration was increased in the zinc supplemented groups in the liver, kidney and lung. The oxygen treatment elevated the metallothionein concentration in the two high zinc supplemented groups (100 and 500 mg Zn/kg diet) in the liver. The response of the zinc concentration in plasma and of hepatic metallothionein levels to oxygen exposure indicates a role of metallothionein in zinc distribution or interactions with other trace elements to support antioxidant capacity, rather than an impact on direct scavenging activity of free radicals.     

Ceruloplasmin and metallothionein induction by zinc and 13-cis-retinoic acid in rats with adjuvant inflammation

The induction of ceruloplasmin and metallothionein was investigated in rats with the early inflammatory phase of adjuvant arthritis. When examined at the peak of the acute inflammatory response, 5 days after adjuvant treatment, zinc given daily (2 mg/kg, intraperitoneally) increased serum ceruloplasmin levels by 2.0 times that found in nonarthritic rats and 1.2 times that found in non-zinc-treated arthritic rats. 13-cis-Retinoic acid (160 mg/kg, orally) given daily increased serum ceruloplasmin 2.2 and 2.7 times that found in nontreated arthritic rats when given alone and with zinc (2 mg/kg, intraperitoneally), respectively. Reduction in the inflammatory response was measured by weight of the adjuvant-injected paw, 5 days after adjuvant was administered. The reduction in inflammation was 13 and 19-20% for 13-cis-retinoic acid and zinc, respectively, when given alone, and between 26 and 31% when the treatments were combined. Zinc markedly increased liver metallothionein levels whereas 13-cis-retinoic acid was a much less potent inducer of the protein in liver. The results are discussed in light of the probable physiological roles of both ceruloplasmin and metallothionein.     

Adrenalectomy enhances the susceptibility of pancreatic islets to interleukin-1 beta: immunohistochemical study.

To determine the importance of adrenal steroid in the effects of interleukin-1, we investigated changes in the number of islet cells reactive toward antiserum to insulin (anti-Ins) by intraperitoneal administration of recombinant human interleukin-1 beta (IL-1) in intact and adrenalectomized (ADX) rats. IL-1 significantly reduced serum insulin levels in ADX rats only, while it similarly decreased plasma glucose levels. In intact rats, IL-1 did not affect the number of islet cells reactive to anti-Ins, although cytoplasmic immunostaining tended to be reduced by IL-1 treatment. Only adrenalectomy decreased the number of islet cells immunostained by anti-Ins. Furthermore, IL-1 treatment significantly reduced the number of islet cells reactive to anti-Ins in ADX rats. The present study immunohistochemically supported our working hypothesis that the withdrawal of adrenal steroids by adrenalectomy enhances the islet cell sensitivity to exogenous administration of IL-1.     

The levels of liver metallothionein and zinc in plaice, Pleuronectes platessa L., during the breeding season, and the effect of oestradiol injection

Wild plaice from two locations were examined for liver metallothionein and liver and serum zinc content, before, during and after the breeding season. During the early stages a number of females had very high liver metallothionein and zinc levels. Egg formation and ripening were accompanied by a reduction in serum zinc. In males, the metallothionein levels did not reach such high values and were not correlated with gonad development.
There was a correlation between the zinc concentration of the liver and the metallothionein concentration of the liver. Above the threshold value for metallothionein formation, approximately half the additional zinc was found in the metallothionein and half in non-metallothionein pool(s).
Intramuscular injection of oestradiol 3-benzoate into immature females caused a significant increase in liver weight but a depression in metallothionein concentration relative to the controls.     

Metallothionein synthesis in foetal, neonatal and maternal rat liver

The synthesis of hepatic metallothionein relative to other cytosol proteins was measured by [35S]cysteine incorporation in foetal, neonatal and pregnant rats. The relative rate of hepatic metallothionein synthesis reached a maximum in foetal liver on days 18-21 of gestation. Metallothionein synthesis then declined until weaning, when adult levels were established. The rate of metallothionein synthesis was greater in pregnant rats at term than in nulliparous rats. To determine if circulating inducing agents could play a role in the regulation of metallothionein synthesis in foetal liver we treated pregnant rats with inducers at a time prior to the normal rise in foetal liver metallothionein synthesis. Injections of copper, cadmium or hydrocortisone to 17-day-pregnant dams failed to induce foetal metallothionein synthesis. In contrast, zinc injection to the dam was an effective inducer in the foetuses. Maternal laparotomy (performed to expose the foetus for direct injection of inducers) induced foetal metallothionein synthesis. Metallothionein synthesis in the livers of 17-day-gestation dams was induced by all metal injections and laparotomy but, surprisingly, not by hydrocortisone injection. Maternal adrenalectomy did not influence the subsequent normal elevation in foetal or maternal metallothionein synthesis. These results, in conjunction with previous reports, suggest that mobilization of zinc in serum during late gestation may regulate foetal and maternal changes in metallothionein synthesis.     

锌对体外应激海马神经元MTs亚型表达的影响

目的:观察不同锌水平对体外应激海马神经元金属硫蛋白(MTs)亚型表达的影响。方法:取新生1dWis-tar大鼠海马组织进行体外神经元培养,无血清培养24h后,分别向培养液中加入皮质酮、Zn2+特异鳌合剂TPEN,使二者的最终浓度均为1×10-5mol/L,然后加入不同浓度的ZnSO4溶液,使Zn2+的最终浓度分别为1×10-5mol/L、1×10-4mol/L和2×10-4mol/L,作用24h后检测培养液中IL-6和NO含量,以蛋白印迹法检测细胞MTs含量,以RT-PCR检测细胞MT-1mRNA和MT-3mRNA的表达水平。结果:在海马神经元培养液中加入TPEN后,MTs的表达出现明显降低,皮质酮刺激也未见其表达升高。在补锌组,MTs的含量均明显增加,其中以10-4mol/LZn2+组的表达量最高。海马神经元MT-1mRNA和MT-3mRNA的表达水平在皮质酮应激组和补锌组均出现明显升高。另外,锌缺乏和皮质酮刺激可使海马神经元培养上清中的IL-6和NO水平均出现明显升高。结论:不同锌水平对应激海马神经元金属硫蛋白及其亚型mRNA的表达具有调控作用,缺锌可降低金属硫蛋白的表达,而补锌可增加金属硫蛋白的表达。     

The influence of restraint stress in rats on metallothionein production and corticosterone and glucagon secretion

Earlier studies on metallothionein (MT) induction by stress used a wide range of stimuli, all of them considered as physical stressors. The present paper reports the effect of a basically psychogenic stress such as restraint on serum and liver MT. Male adult rats were stressed for 1, 12, 24 and 48 hours and then killed. Liver MT increased continuously throughout the experimental period. Rats deprived of water and food for 24 and 48 hours showed higher liver MT levels than control but lower levels than stressed ones. Serum MT was not modified by either restraint or water and food deprivation. The lack of relationship between the two MT pools was corroborated by the absence of a significant correlation between them. Restraint stress increased serum corticosterone but not glucagon levels, suggesting that only glucocorticoids are important in MT induction by stress. However, a strong circadian rhythmicity was observed in serum corticosterone but not in serum or liver MT in non-stressed rats. In addition, preliminary data with adrenalectomized rats indicate that glucocorticoids seem not to be essential in MT induction by stress. Likewise glucagon does not appears to be associated with MT induction by stress since its levels were not modified by restraint.     

Induction of hepatic metallothionein by salicylate in adult rats

Application of salicylate increased the concentration of metallothionein (MT) in liver of pregnant rats as well as of adult male rats, whereas in fetal liver, MT was reduced by salicylate. Induction of MT synthesis by salicylate is an indirect effect because in cultured hepatocytes salicylate did not induce MT synthesis. Salicylate increased MT also in adrenalectomized rats. Indomethacin induced the same concentration of MT in maternal liver as salicylate. However, indomethacin had no effect on MT in fetal liver. Induction of MT in adult liver by salicylate and indomethacin was independent of zinc.     

Zinc Supplementation Attenuates Metallothionein and Oxidative Stress Changes in Kidney of Streptozotocin-Induced Diabetic Rats

Zinc is an element that under physiological conditions preferentially binds to and is a potent inducer of metallothionein under physiological conditions. The present study was conducted to explore whether zinc supplementation morphologically and biochemically protects against diabetic nephropathy through modulation of kidney metallothionein induction and oxidative stress in streptozotocin-induced diabetic rats. Thirty-two Wistar albino male rats were equally divided into four groups. The first group was used as untreated controls and the second group was supplemented with 30?mg/kg/day zinc as zinc sulfate. The third group was treated with streptozotocin to induce diabetes and the fourth group was treated with streptozotocin and supplemented with zinc as described for group 2. The blood glucose and micro-albuminuria levels, body and kidney weights were measured during the 42-day experimental period. At the end of the experiment, the kidneys were removed from all animals from the four groups. Diabetes resulted in degenerative kidney morphological changes. The metallothionein immunoreactivity level was lower and the kidney lipid peroxidation levels were higher in the diabetes group than in the controls. The metallothionein immunoreactivity levels were higher in the tubules of the zinc-supplemented diabetic rats as compared to the non-supplemented diabetic group. The zinc and metallothionein concentrations in kidney tissue were higher in the supplemented diabetic group compared to the non-supplemented diabetes group. The activity of glutathione peroxidase did not change in any of the four groups. In conclusion, the present study shows that zinc has a protective effect against diabetic damage of kidney tissue through stimulation of metallothionein synthesis and regulation of the oxidative stress.