To characterize the physiological role of metallothioneins I and II (MT-I+II) in the brain, we have examined the chronological effects of a freeze injury to the cortex in normal and MT-I+II null mice. In normal mice, microglia/macrophage activation and astrocytosis were observed in the areas surrounding the lesion site, peaking at approximately 1 and 3 d postlesion (dpl), respectively. At 20 dpl, the parenchyma had regenerated. Both brain macrophages and astrocytes surrounding the lesion increased the MT-I+II immunoreactivity, peaking at approximately 3 dpl, and at 20 dpl it was similar to that of unlesioned mice. In situ hybridization analysis indicates that MT-I+II immunoreactivity reflects changes in the messenger levels. In MT-I+II null mice, microglia/macrophages infiltrated the lesion heavily, and at 20 dpl they were still present. Reactive astrocytosis was delayed and persisted at 20 dpl. In contrast to normal mice, at 20 dpl no wound healing had occurred. The rate of apoptosis, as determined by using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling, was drastically increased in neurons of ipsilateral cortex of the MT-I+II null mice. Our results demonstrate that MT-I+II are essential for a normal wound repair in the CNS, and that their deficiency impairs neuronal survival.

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We examined metallothionein (MT)-induced neuroprotection during kainic acid (KA)-induced excitotoxicity by studying transgenic mice with MT-I overexpression (TgMT mice). KA induces epileptic seizures and hippocampal excitotoxicity, followed by inflammation and delayed brain damage. We show for the first time that even though TgMT mice were more susceptible to KA, the cerebral MT-I overexpression decreases the hippocampal inflammation and delayed neuronal degeneration and cell death as measured 3 days after KA administration. Hence, the proinflammatory responses of microglia/macrophages and lymphocytes and their expression of interleukin (IL)-1, IL-6, IL-12, tumor necrosis factor-alpha and matrix metalloproteinases (MMP-3, MMP-9) were significantly reduced in hippocampi of TgMT mice relative to wild-type mice. Also by 3 days after KA, the TgMT mice showed significantly less delayed damage, such as oxidative stress (formation of nitrotyrosine, malondialdehyde, and 8-oxoguanine), neurodegeneration (neuronal accumulation of abnormal proteins), and apoptotic cell death (judged by TUNEL and activated caspase-3). This reduced bystander damage in TgMT mice could be due to antiinflammatory and antioxidant actions of MT-I but also to direct MT-I effects on the neurons, in that significant extracellular MT presence was detected. Furthermore, MT-I overexpression stimulated astroglia and increased immunostaining of antiinflammatory IL-10, growth factors, and neurotrophins (basic fibroblastic growth factor, transforming growth factor-beta, nerve growth factor, brain-derived neurotrophic factor, glial-derived neurotrophic factor) in hippocampus. Accordingly, MT-I has different functions that likely contribute to the increased neuron survival and improved CNS condition of TgMT mice. The data presented here add new insight into MT-induced neuroprotection and indicate that MT-I therapy could be used against neurological disorders.

Interleukin (IL)-6 is a pleiotropic hormone that has both proinflammatory and anti-inflammatory actions. AMP-activated protein kinase (AMPK) is a fuel-sensing enzyme that among its other actions responds to decreases in cellular energy state by enhancing processes that generate ATP and inhibiting others that consume ATP but are not acutely necessary for survival. IL-6 is synthesized and released from skeletal muscle in large amounts during exercise, and in rodents, the resultant increase in its concentration correlates temporally with increases in AMPK activity in multiple tissues. That IL-6 may be responsible in great measure for these increases in AMPK is suggested by the fact it increases AMPK activity both in muscle and adipose tissue in vivo and in incubated muscles and cultured adipocytes. In addition, we have found that AMPK activity is diminished in muscle and adipose tissue of 3-month-old IL-6 knockout (KO) mice at rest and that the absolute increases in AMPK activity in these tissues caused by exercise is diminished compared with control mice. Except for an impaired ability to exercise and to oxidize fatty acids, the IL-6 KO mouse appears normal at 3 months of age. On the other hand, by age 9 months, it manifests many of the abnormalities of the metabolic syndrome including obesity, dyslipidemia, and impaired glucose tolerance. This, plus the association of decreased AMPK activity with similar abnormalities in a number of other rodents, suggests that a decrease in AMPK activity may be a causal factor. Whether increases in IL-6, by virtue of their effects on AMPK, contribute to the reported ability of exercise to diminish the prevalence of type 2 diabetes, coronary heart disease, and other disorders associated with the metabolic syndrome remains to be determined. Diabetes 55 (Suppl. 2):S48 -S54, 2006

Summary:To study the importance of metallothionein-I and -II (MT-I+II) for brain inflammation and regeneration, the authors examined normal and MT-I+II knock-out (MT-KO) mice subjected to a cortical freeze injury. Normal mice showed profound neurodegeneration, inflammation, and gliosis around the injury, which was repaired by 20 days postlesion (dpl). However, in MT-KO mice the lesion-associated inflammation was still present as late as 90 dpl. Scanning electron microscopy demonstrated that the number of capillaries was lower, and ultrastructural preservation of the lesioned parenchyma was poorer in MT-KO mice, suggesting an altered angiogenesis. To gain insight into the mechanisms involved, a number of cytokines and growth factors were evaluated. The number of cells expressing the proinflammatory cytokines IL-1␤, IL-6, and TNF-␣ was higher in MT-KO mice than in normal mice, which was confirmed by RNase protection analysis, whereas the number of cells expressing the growth factors bFGF, TGF␤1, VEGF, and NT-3 was lower. Increased expression of proinflammatory cytokines could be involved in the sustained recruitment of CD-14+ and CD-34+ inflammatory cells and their altered functions observed in MT-KO mice. Decreases in trophic factors bFGF, TGF␤1, and VEGF could mediate the decreased angiogenesis and regeneration observed in MT-KO mice after the freeze lesion. A role for MT-I+II in angiogenesis was also observed in transgenic mice expressing IL-6 under the control of the promoter of glial fibrillary acidic protein gene (GFAP-IL6 mice) because MT-I+II deficiency dramatically decreased the IL-6-induced angiogenesis of the GFAP-IL6 mice. In situ hybridization analysis indicated that the MT-III expression was not altered by MT-I+II deficiency. These results suggest that the MT-I+II isoforms have major regulatory functions in the brain inflammatory response to injury, especially in the angiogenesis process. Key Words: MT-I+II-MT-IIIInflammation-Angiogenesis-Regeneration-Cytokines.Metallothioneins are a family of cysteine-rich, ubiquitous intracellular proteins. In the central nervous system (CNS), MT-I+II are expressed coordinately and are increased in macrophages/microglia and astrocytes during various inflammatory and pathologic conditions, including human neurodegenerative disorders, experimentally induced brain injury, epileptic seizures, brain ischemia, astroglial cell death, metal exposure, stress, and myelin deficiency