Effect of dietary zinc deficiency on brain metallothionein-I and -III mRNA levels during stress and inflammation (original) (raw)
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Journal of Neurotrauma, 2001
The role of zinc-and copper-deficient diets on the inflammatory response to traumatic brain injury (TBI) has been evaluated in adult rats. As expected, zinc deficiency decreased food intake and body weight gain, and the latter effect was higher than that observed in pair-fed rats. In noninjured brains, zinc deficiency only affected significantly lectin (increasing) and glial fibrillary acidic protein (GFAP) and Cu,Zn-superoxide dismutase (Cu,Zn-SOD) (decreasing) immunoreactivities (irs). In injured brains, a profound gliosis was observed in the area surrounding the lesion, along with severe damage to neurons as indicated by neuron specific enolase (NSE) ir, and the number of cells undergoing apoptosis (measured by TUNEL) was dramatically increased. Zinc deficiency significantly altered brain response to TBI, potentiating the microgliosis and reducing the astrogliosis, while increasing the number of apoptotic cells. Metallothioneins (MTs) are important zinc-and copper-binding proteins in the CNS, which could influence significantly the brain response to TBI because of their putative roles in metal homeostasis and antioxidant defenses. MT-I1 II expression was dramatically increased by TBI, and this response was significantly blunted by zinc deficiency. The MT-III isoform was moderately increased by both TBI and zinc deficiency. TBI strongly increased oxidative stress levels, as demonstrated by malondialdehyde (MDA), protein tyrosine nitration (NITT), and nuclear factor kB (NF-kB) levels irs, all of which were potentiated by zinc deficiency. Further analysis revealed unbalanced expression of prooxidant and antioxidant proteins besides MT, since the levels of inducible nitric oxide synthase (iNOS) and Cu,Zn-SOD were increased and decreased, respectively, by zinc deficiency. All these effects were attributable to zinc deficiency, since pair-fed rats did not differ from normally fed rats. In general, copper deficiency caused a similar pattern of responses, albeit more moderate. Results obtained in mice with a null mutation for the MT-I1 II isoforms strongly suggest that most of the effects observed in the rat brain after zinc and copper deficiencies are attributable to the concomitant changes in the MT expression.
Regulation of zinc metallothionein II mRNA level in rat brain
Neurochemistry International, 1990
Akstract--Metallothionein isoforms I and II (MTI and MTII) have been identified in the rat brain, monkey brain, bovine retina, pineal gland and hippocampus, and in the neuroblastoma IMR 32. Since intraperitoneally administered zinc passes across the blood brain barrier slowly, the rat brain metallothionein can be induced in a time-and dose-dependent fashion only following intracerebroventricularly (i.c.v.) administered zinc sulfate at a rate of 0.20 #mol/#l/h for 24 h using an Alzet minipump. The zinc-induced proteins, incorporate large quantities of [35S]cysteine, bind 65Zn and produce two isoforms which contain 17 and 18 cysteine residues, respectively, but lack aromatic amino acids or histidine. In this communication, we report that i.c.v.-administered zinc in a bolus of 0.1 and 0.5 #mol increased the synthesis of poly A ÷ RNA from 6.6 to 8.0 and 9.6 #g/g brain tissue, respectively. Furthermore, we probed the poly A ÷ RNA with 3Zp-labeled 180 base pair BamHl/PvulI restriction fragment containing the cDNA for human MTII from the phMT-II3 plasmid. Slot blot analysis of poly A + RNA revealed a dose-dependent increase in brain MTII hybridizable mRNA. Northern blot analysis of poly A ÷ RNA extracted from the rat liver and brain using 3Zp-labeled cDNA exhibited a major band of hybridization at 700 bases in both tissues. These data provide evidence that the zinc-induced MT synthesis in the brain is associated with an accumulation of mRNA which is analogous to the zinc-induced synthesis of hepatic MT mRNA. However, other evidence indicates that the factors regulating the synthesis of the brain and the hepatic metallothioneins are not identical.
PLoS ONE, 2012
Experiments with transgenic over-expressing, and null mutant mice have determined that metallothionein-I and -II (MT-I/II) are protective after brain injury. MT-I/II is primarily a zinc-binding protein and it is not known how it provides neuroprotection to the injured brain or where MT-I/II acts to have its effects. MT-I/II is often expressed in the liver under stressful conditions but to date, measurement of MT-I/II expression after brain injury has focused primarily on the injured brain itself. In the present study we measured MT-I/II expression in the liver of mice after cryolesion brain injury by quantitative reverse-transcriptase PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) with the UC1MT antibody. Displacement curves constructed using MT-I/II knockout (MT-I/II 2/2 ) mouse tissues were used to validate the ELISA. Hepatic MT-I and MT-II mRNA levels were significantly increased within 24 hours of brain injury but hepatic MT-I/II protein levels were not significantly increased until 3 days post injury (DPI) and were maximal at the end of the experimental period, 7 DPI. Hepatic zinc content was measured by atomic absorption spectroscopy and was found to decrease at 1 and 3 DPI but returned to normal by 7DPI. Zinc in the livers of MT-I/II 2/2 mice did not show a return to normal at 7 DPI which suggests that after brain injury, MT-I/II is responsible for sequestering elevated levels of zinc to the liver. Conclusion: MT-I/II is upregulated in the liver after brain injury and modulates the amount of zinc that is sequestered to the liver.
Metallothionein-I induction by stress in specific brain areas
Neurochemical Research, 1991
The distribution of metallothionein-I (MT) in several areas of the brain and its induction by immobilization stress has been studied in the rat. MT content was highest in hippocampus and midbrain and lowest in frontal cortex and pons plus medulla oblongata. Immobilization stress for 18 hours (which was accompanied by food and water deprivation) significantly increased MT levels in the frontal cortex, pons plus medulla oblongata and hypothalamus, but not in midbrain and hippocampus. The effect of stress on MT levels was specific as food and water deprivation along had no significant effect on MT levels in any of the brain areas studied. The effect of stress on MT levels was independent of changes in cytosolic Zn content; this was generally unaffected by stress or food and water deprivation but decreased in pons plus medulla oblongata from stressed rats. The results suggest that MT is induced more significantly in the brain areas that are usually involved in the response of animals to stress.
Expression and regulation of brain metallothionein
Neurochemistry International, 1995
Many, but not all, zinc-containing neurons in the brain are a subclass of the glutamatergic neurons, and they are found predominantly in the telencephalon. These neurons store zinc in their presynaptic terminals and release it by a calcium-dependent mechanism. These "vesicular" pools of zinc are viewed as endogenous modulators of ligand-and voltage-gated ion channels. Metallothioneins (MTs) are low molecular weight zinc-binding proteins consisting of 25-30% cysteine, with no aromatic amino acids or disulfide bonds. The areas of the brain containing high contents of zinc such as the retina, the pineal gland, and the hippocampus synthesize unique isoforms of MT on a continuous basis. The four MT isoforms are thought to provide the neurons and glial elements with mechanisms to distribute, donate, and sequester zinc at presynaptic terminals; or buffer the excess zinc at synaptic junctions. In this cause, glutathione disulfide may participate in releasing zinc from MT. A similar nucleotide and amino acid sequence has made it difficult to obtain cDNA probes and antibodies capable of distinguishing indisputably among MT isoforms. MT-I and MT-II isoforms are found in the brain and in the peripheral tissues ; MT-III isoform, possessing an additional seven amino acids, is expressed mostly in the brain and to a very minute extent in the intestine and pancreas ; whereas MT-IV isoform is found in tissues containing stratified squamous epithelial cells, Since MTs are expressed in neurons that sequester zinc in their synaptic vesicles, the regulation of the expression of MT isoforms is extremely important in terms of maintaining the steadystate level of zinc and controlling redox potentials. The concentration of zinc has been shown to be altered in an extensive number of disorders of the central nervous system, including alcoholism, Alzheimer-type dementia, amyotrophic lateral sclerosis, Down's syndrome, epilepsy, Friedreich's ataxia, Guillaine-Barr6 syndrome, hepatic encephalopathy, multiple sclerosis, Parkinson's disease, Pick's disease, retinitis pigmentosa, retinal dystrophy, schizophrenia, and Wernicke-Korsakoff syndrome. The status of MT isoforms and other low molecular weight zinc-binding proteins in these conditions, diseases, disorders, or syndromes is being delineated at this time. Since several of these disorders, such as amyotrophic lateral sclerosis, are associated with oxidative stress, and since MT is able to prevent the formation of free radicals, it is believed that cytokine-induced induction of MT provides a long-lasting protection to avert oxidative damage.
Hepatic Responses to Dietary Stress in Zinc and Metallothionein-Deficient Mice
Metallothionein (MT) and zinc are both reported to be protective against oxidative and inflammatory stress and may also influence energy metabolism. The role of MT in regulating intracellular labile zinc, thus influencing zinc (Zn)-modulated protein activity, may be a key factor in the response to stress and other metabolic challenges. The objective of this study was to investigate the influence of dietary zinc intake and MT on hepatic responses to a pro-oxidant stress and energy challenge in the form of a high dietary intake of linoleic acid, an omega-6 polyunsaturated fatty acid. Male MT-null (KO) and wild-type (WT) mice, aged 16 weeks, were given semisynthetic diets containing 16% fat and either 5 (marginally zinc-deficient [ZD]) or 35 (zincadequate [ZA]) mg Zn/kg. For comparison, separate groups of KO and WT mice were given a rodent chow diet containing 3.36% fat and 86.6 mg Zn/kg. After 4 months on these diets, the body weights of all mice were equal, but liver size, weight, and lipid content were much greater in the animals that consumed semisynthetic diets compared to the chow diet. The increase in liver size was significantly lower in ZA but not ZD KO mice, compared with WT mice. Principally, MT appears to affect the diet-induced increase in liver tissue but it also influences the concentration of hepatic lipid. Plasma levels of C-reactive protein (CRP), a marker of inflammation, were increased by zinc deficiency in WT mice, suggesting that marginal zinc deficiency is proinflammatory. CRP was unaffected by zinc deficiency in KO mice, indicating a role for MT in modulating the influence of zinc. Neither zinc nor MT deficiency affects the level of soluble liver proteins, as determined using two-dimensional (2D) gel proteomics. This study highlights the close association between zinc and MT in the manifestation of stress responses.
Chemico-biological Interactions, 1994
The knowledge of brain metallothlonem (MT) regulation and especially of MT presence in specific cell types Is scarce Therefore, the effect of several well-known MT reducers, measured by ra&olmmunoassays using antlbo&es that cross-react with MT-I and MT-II or specific for MT-I and which do not cross-react with human growth inhibitory factor (GIF or MT-III), has been stu&ed m primary cultures of neurons or astrocytes obtained from rat cerebrum MT-I levels m ghal cells were about ten times higher than those m neuronal cells (538 ± 194 vs 49 ± 16 pg MT-I/t~g protein, mean ± S D from three separate cell preparations) Increas-Ing the concentration of Zn in the bowne serum albumm (BSA)-contammg culture medmm up to 50/~M slgmficantly increased MT-I levels by up to 3 5-fold in neurons and 2 5-fold in astrocytes In contrast, Cu up to 50 #M increased MT-I levels m a saturable manner in both neurons (up to 5-fold) and astrocytes (up to 1 5-fold), the maximum effect occurring at 5 #M Cu. In general, the combination of Zn and Cu further increased MT-I levels The effect of the metals on MT-I appeared to reflect metal uptake, since MT-I induction was less marked when the BSA concentration in the medium was increased from 2 to 10 mg/ml Dexamethasone increased MT-I levels in both neurons and astrocytes in vitro in a concentrationdependent manner Endotoxln, IL-l and IL-6 did not have a significant effect on ghal MT levels at the concentrations studied The administration of dexamethasone to rats increased MT-I levels m non-frontal cortex, cerebellum, pons + medulla, mldbraln and hlppocampus, but not m hypothalamus, frontal cortex and strlatum Endotoxln increased liver but not brain MT-I levels Immunocytochemlcal studies in adult rat brain preparations with a polyclonal antibody that cross-reacts with MT-I and MT-II indicated that immunostalnlng was always nuclear in ghal cells, whereas in neurons it was nuclear in the cerebral cortex, hlppocampus and the granular layer of the cerebellum, and nuclear plus cytoplasmic in Purklnje cells in the cerebellum, hypothalamlc nuclei and glgantocellular reticular nucleus in the brain stem Menlnges, choroldal plexus, ependymal and endothehal cells were also MT-lmmunoreactlve
Distribution of zinc metallothionein I mRNA in rat brain using in situ hybridization
Neurochemical Research, 1994
Metallothionein (MT) isoforms ! and II were first identified and characterized in our laboratories in several regions of brain, in hippocampal neurons in primary cuIture, and in retinoblastoma and neuroblastoma cell lines. In this study, by having employed the MT-I cDNA as a probe, we sought to gain additional insight about the function of MT by discerning the regional distribution of its mRNA. Northern blot analyses of brain mRNA revealed that the administration of zinc enhanced dramatically MT-I mRNA (570 bp). The in situ hybridization study revealed that MT-I mRNA was located in several areas of brain, with the highest concentrations found in the cerebellum, hippocampus, and ventricles. The results of these studies are interpreted to suggest that zinc enhances the synthesis of MT mRNA and MT in turn may participate in zinc associated functions in neurons.
Metallothionein-I+II induction by zinc and copper in primary cultures of rat microglia
Neurochemistry International, 1998
Metallothioneins "MTs# are a family of low molecular weight proteins which in rodents comprise four isoforms "MT!I to !IV#[ MT!I¦II are widely expressed isoforms which are highly inducible by factors such as heavy metals and a number of hormones[ The expression of these isoforms in the brain has been regarded as basically astrocytic\ and to a lower extent\ neuronal[ We\ however\ demonstrate in this report\ by radioimmunoassay and immunocytochemistry\ that MT!I¦II are expressed in primary cultures of rat microglia and that\ furthermore\ they are inducible by zinc and copper[ Thus all major brain cell types may express the MT!I¦II isoforms and respond with increased protein levels to physiological stimuli such as increased extracellular zinc and copper content[ In contrast\ microglia MT!I¦II levels are not a}ected by either the glucocorticoid dexamethasone or the cytokine IL!0 under the experimental conditions[ Þ 0887 Elsevier Science Ltd[ All rights reserved[ Corresponding author[ Tel[ 99232 470 1926^fax] 99232 470 1289ê !mail] HidalgoÝcc[uab[es
Zinc Metabolism in the Brain: Relevance to Human Neurodegenerative Disorders
Neurobiology of Disease, 1997
Zinc is an important trace element in biology. An important pool of zinc in the brain is the one present in synaptic vesicles in a subgroup of glutamatergic neurons. In this form it can be released by electrical stimulation and may serve to modulate responses at receptors for a number of different neurotransmitters. These include both excitatory and inhibitory receptors, particularly the NMDA and GABA(A) receptors. This pool of zinc is the only form of zinc readily stained histochemically (the chelatable zinc pool), but constitutes only about 8% of the total zinc content in the brain. The remainder of the zinc is more or less tightly bound to proteins where it acts either as a component of the catalytic site of enzymes or in a structural capacity. The metabolism of zinc in the brain is regulated by a number of transport proteins, some of which have been recently characterized by gene cloning techniques. The intracellular concentration may be mediated both by efflux from the cell by the zinc transporter ZrT1 and by complexing with apothionein to form metallothlonein. Metallothionein may serve as the source of zinc for incorporation into proteins, including a number of DNA transcription factors. However, zinc is readily released from metallothionein by disulfides, increasing concentrations of which are formed under oxidative stress. Metallothionein is a very good scavenger of free radicals, and zinc itself can also reduce oxidative stress by binding to thiol groups, decreasing their oxidation. Zinc is also a very potent inhibitor of nitric oxide synthase. Increased levels of chelatable zinc have been shown to be present in cell cultures of immune cells undergoing apoptosis. This is very reminiscent of the zinc staining of neuronal perikarya dying after an episode of ischemia or seizure activity. Thus a possible role of zinc in causing neuronal death in the brain needs to be fully investigated. intraventricular injections of calcium EDTA have already been shown to reduce neuronal death after a period of ischemia. Pharmacological doses of zinc cause neuronal death, and some estimates indicate that extracellular concentrations of zinc could reach neurotoxic levels under pathological conditions. Zinc is released in high concentrations from the hippocampus during seizures. Unfortunately, there are contrasting observations as to whether this zinc serves to potentiate or decrease seizure activity. Zinc may have an additional role in causing death in at least some neurons damaged by seizure activity and be involved in the sprouting phenomenon which may give rise to recurrent seizure propagation in the hippocampus. In Alzheimer's disease, zinc has been shown to aggregate beta-amyloid, a form which is potentially neurotoxic. The zinc-dependent transcription factors NF-kappa B and Sp1 bind to the promoter region of the amyloid precursor protein (APP) gene. Zinc also inhibits enzymes which degrade APP to nonamyloidogenic peptides and which degrade the soluble form of beta-amyloid. The changes in zinc metabolism which occur during oxidative stress may be important in neurological diseases where oxidative stress is implicated, such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS). Zinc is a structural component of superoxide dismutase 1, mutations in which give rise to one form of familiar ALS. After HIV infection, zinc deficiency is found which may be secondary to immune-induced cytokine synthesis. Zinc is involved in the replication of the HIV virus at a number of sites. These observations should stimulate further research into the role of zinc in neuropathology.