Milena Penkowa

Brain-derived neurotrophic factor is produced by skeletal muscle cells in response to contraction and enhances fat oxidation via activation of AMP-activated protein kinase

It has been brought to our attention following an investigation into the work of Bente Klarlund Pedersen by the Danish Committees for Scientific Dishonesty, that the erratum published in 2012 was insufficient to correct this article. Although the data published in the Diabetologia paper were previously unpublished, the data from the biological material collected from the additional eight healthy men presented in Fig. 1b and c originated from a previous study that was not referenced [1]. In addition, while these eight healthy subjects performed the same type of exercise at the same intensity, the duration was different. The following description of the methodology and Fig. 1 legend correct these oversights. The authors would like to reiterate that these methodological oversights in no way affect either the data presented in the paper or the conclusions reached. The authors also apologise to both the journal and its readership for these oversights.

Supplementation with vitamins C and E inhibits the release of interleukin‐6 from contracting human skeletal muscle

Contracting human skeletal muscle is a major contributor to the exercise‐induced increase of plasma interleukin‐6 (IL‐6). Although antioxidants have been shown to attenuate the exercise‐induced increase of plasma IL‐6, it is unknown whether antioxidants inhibit transcription, translation or translocation of IL‐6 within contracting human skeletal muscle. Using a single‐blind placebo‐controlled design with randomization, young healthy men received an oral supplementation with either a combination of ascorbic acid (500 mg day−1) and RRR‐α‐tocopherol (400 i.u. day−1) (Treatment, n= 7), or placebo (Control, n= 7). After 28 days of supplementation, the subjects performed 3 h of dynamic two‐legged knee‐extensor exercise at 50% of their individual maximal power output. Muscle biopsies from vastus lateralis were obtained at rest (0 h), immediately post exercise (3 h) and after 3 h of recovery (6 h). Leg blood flow was measured using Doppler ultrasonography. Plasma IL‐6 concentration was measured in blood sampled from the femoral artery and vein. The net release of IL‐6 was calculated using Ficks principle. Plasma vitamin C and E concentrations were elevated in Treatment compared to Control. Plasma 8‐iso‐prostaglandin F2α, a marker of lipid peroxidation, increased in response to exercise in Control, but not in Treatment. In both Control and Treatment, skeletal muscle IL‐6 mRNA and protein levels increased between 0 and 3 h. In contrast, the net release of IL‐6 from the leg, which increased during exercise with a peak at 3.5 h in Control, was completely blunted during exercise in Treatment. The arterial plasma IL‐6 concentration from 3 to 4 h, when the arterial IL‐6 levels peaked in both groups, was ∼50% lower in the Treatment group compared to Control (Treatment versus Control: 7.9 pg ml−1, 95% confidence interval (CI) 6.0–10.7 pg ml−1, versus 19.7 pg ml−1, CI 13.8–29.4 pg ml−1, at 3.5 h, P < 0.05 between groups). Moreover, plasma interleukin‐1 receptor antagonist (IL‐1ra), C‐reactive protein and cortisol levels all increased after the exercise in Control, but not in Treatment. In conclusion, our results show that supplementation with vitamins C and E attenuated the systemic IL‐6 response to exercise primarily via inhibition of the IL‐6 protein release from the contracting skeletal muscle per se.

Strongly compromised inflammatory response to brain injury in interleukin‐6‐deficient mice

Injury to the central nervous system (CNS) elicits an inflammatory response involving activation of microglia, brain macrophages, and astrocytes, processes likely mediated by the release of proinflammatory cytokines. In order to determine the role of interleukin‐6 (IL‐6) during the inflammatory response in the brain following disruption of the blood–brain barrier (BBB), we examined the effects of a focal cryo injury to the fronto‐parietal cortex in interleukin‐6‐deficient (IL‐6−/−) and normal (IL‐6+/+) mice. In IL‐6+/+ mice, brain injury resulted in the appearance of brain macrophages and reactive astrocytes surrounding the lesion site. In addition, expression of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and metallothionein‐I+II (MT‐I+II) were increased in these cells, while the brain‐specific MT‐III was only moderately upregulated. In IL‐6−/− mice, however, the response of brain macrophages and reactive astrocytes was markedly depressed and the number of NSE positive neurons was reduced. Brain damage‐induced GM‐CSF and MT‐I+II expression were also markedly depressed compared to IL‐6+/+ mice. In contrast, MT‐III immunoreactivity was markedly increased in brain macrophages and astrocytes. In situ hybridization analysis indicates that MT‐I+II but not MT‐III immunoreactivity reflect changes in the messenger levels. The number of cell divisions was similar in IL‐6+/+ and IL‐6−/− mice. The present results demonstrate that IL‐6 is crucial for the recruitment of myelo‐monocytes and activation of glial cells following brain injury with disrupted BBB. Furthermore, our results suggest IL‐6 is important for neuroprotection and the induction of GM‐CSF and MT expression. The opposing effect of IL‐6 on MT‐I+II and MT‐III levels in the damaged brain suggests MT isoform‐specific functions. GLIA 25:343–357, 1999.

Expression of interleukin-15 in human skeletal muscle – effect of exercise and muscle fibre type composition

The cytokine interleukin‐15 (IL‐15) has been demonstrated to have anabolic effects in cell culture systems. We tested the hypothesis that IL‐15 is predominantly expressed by type 2 skeletal muscle fibres, and that resistance exercise regulates IL‐15 expression in muscle. Triceps brachii, vastus lateralis quadriceps and soleus muscle biopsies were obtained from normally physically active, healthy, young male volunteers (n= 14), because these muscles are characterized by having different fibre‐type compositions. In addition, healthy, normally physically active male subjects (n= 8) not involved in any kind of resistance exercise underwent a heavy resistance exercise protocol that stimulated the vastus lateralis muscle and biopsies were obtained from this muscle pre‐exercise as well as 6, 24 and 48 h post‐exercise. IL‐15 mRNA levels were twofold higher in the triceps (type 2 fibre dominance) compared with the soleus muscle (type 1 fibre dominance), but Western blotting and immunohistochemistry revealed that muscle IL‐15 protein content did not differ between triceps brachii, quadriceps and soleus muscles. Following resistance exercise, IL‐15 mRNA levels were up‐regulated twofold at 24 h of recovery without any changes in muscle IL‐15 protein content or plasma IL‐15 at any of the investigated time points. In conclusion, IL‐15 mRNA level is enhanced in skeletal muscles dominated by type 2 fibres and resistance exercise induces increased muscular IL‐15 mRNA levels. IL‐15 mRNA levels in skeletal muscle were not paralleled by similar changes in muscular IL‐15 protein expression suggesting that muscle IL‐15 may exist in a translationally inactive pool.

Immunohistochemical detection of interleukin-6 in human skeletal muscle fibers following exercise

Interleukin‐6 (IL‐6) is produced by many different cell types. Human skeletal muscles produce and release high amounts of IL‐6 during exercise; however, the cell source of origin in the muscle is not known. Therefore, we studied the protein expression of IL‐6 by immunohistochemistry in human muscle tissue from biopsies obtained at time points 0, 3, 4.5, 6, 9, and 24 h in relation to 3 h of bicycle exercise performed by healthy young males (n=12) and in resting controls (n=6). The IL‐6 expression was clearly increased after exercise and remained high even by 24 h, relative to pre‐exercise or resting individuals. The IL‐6 immunostainings of skeletal muscle cells were homogeneous and without difference between muscle fiber types. The IL‐6 mRNA peaked immediately after the exercise, and, in accordance, the IL‐6 protein expression within muscle cells was most pronounced around 3 h post‐exercise. However, the finding that plasma IL‐6 concentration peaked in the end of exercise indicates a high turnover of muscle‐derived IL‐6. In conclusion, the finding of marked IL‐6 protein expression exclusively within skeletal muscle fibers following exercise demonstrates that skeletal muscle fibers of all types are the dominant cell source of exercise‐induced release of IL‐6 from working muscle.

Impaired inflammatory response and increased oxidative stress and neurodegeneration after brain injury in interleukin-6-deficient mice.

In order to determine the role of the neuropoietic cytokine interleukin‐6 (IL‐6) during the first 3 weeks after a focal brain injury, we examined the inflammatory response, oxidative stress and neuronal survival in normal and interleukin‐6‐deficient (knockout, IL‐6KO) mice subjected to a cortical freeze lesion. In normal mice, the brain injury was followed by reactive astrogliosis and recruitment of macrophages from 1 day postlesion (dpl), peaking at 3–10 dpl, and by 20 dpl the transient immunoreactions were decreased, and a glial scar was present. In IL‐6KO mice, the reactive astrogliosis and recruitment of macrophages were decreased throughout the experimental period. The expression of the antioxidant and anti‐apoptotic factors metallothionein I+II (MT‐I+II) was increased prominently by the freeze lesion, but this response was significantly reduced in the IL‐6 KO mice. By contrast, the expression of the antioxidants Cu/Zn‐superoxide dismutase (Cu/Zn‐SOD), Mn‐SOD, and catalase remained unaffected by the IL‐6 deficiency. The lesioned mice showed increased oxidative stress, as judged by malondialdehyde (MDA) and nitrotyrosine (NITT) levels and by formation of inducible nitric oxide synthase (iNOS). IL‐6KO mice showed higher levels of MDA, NITT, and iNOS than did normal mice. Concomitantly, in IL‐6KO mice the number of apoptotic neurons was significantly increased as judged by TUNEL staining, and regeneration of the tissue was delayed relative to normal mice. The changes in neuronal tissue damage and in brain regeneration observed in IL‐6KO mice are likely caused by the IL‐6‐dependent decrease in MT‐I+II expression, indicating IL‐6 and MT‐I+II as neuroprotective factors during brain injury. GLIA 32:271–285, 2000.

The role of metallothionein in oncogenesis and cancer prognosis.

The antiapoptotic, antioxidant, proliferative, and angiogenic effects of metallothionein (MT)-I+II has resulted in increased focus on their role in oncogenesis, tumor progression, therapy response, and patient prognosis. Studies have reported increased expression of MT-I+II mRNA and protein in various human cancers; such as breast, kidney, lung, nasopharynx, ovary, prostate, salivary gland, testes, urinary bladder, cervical, endometrial, skin carcinoma, melanoma, acute lymphoblastic leukemia (ALL), and pancreatic cancers, where MT-I+II expression is sometimes correlated to higher tumor grade/stage, chemotherapy/radiation resistance, and poor prognosis. However, MT-I+II are downregulated in other types of tumors (e.g. hepatocellular, gastric, colorectal, central nervous system (CNS), and thyroid cancers) where MT-I+II is either inversely correlated or unrelated to mortality. Large discrepancies exist between different tumor types, and no distinct and reliable association exists between MT-I+II expression in tumor tissues and prognosis and therapy resistance. Furthermore, a parallel has been drawn between MT-I+II expression as a potential marker for prognosis, and MT-I+IIs role as oncogenic factors, without any direct evidence supporting such a parallel. This review aims at discussing the role of MT-I+II both as a prognostic marker for survival and therapy response, as well as for the hypothesized role of MT-I+II as causal oncogenes.

Metallothionein-1+2 protect the CNS after a focal brain injury

We have evaluated the physiological relevance of metallothionein-1+2 (MT-1+2) in the CNS following damage caused by a focal cryolesion onto the cortex. In comparison to normal mice, transgenic mice overexpressing the MT-1 isoform (TgMTI* mice) showed a significant decrease of the number of activated microglia/macrophage and of CD3+ T lymphocytes in the area surrounding the lesion, while astrocytosis was increased. The TgMTI* mice showed a diminished peripheral macrophage but not CD3 T cell response to the cryolesion. This altered inflammatory response produced a decreased expression of the proinflammatory cytokines IL-1beta, IL-6, and TNF-alpha and an increased expression of the growth factors bFGF, TGFbeta1, and VEGF in the TgMTI* mice relative to control mice, which might be related to the increased angiogenesis and regeneration of the parenchyma of the former mice. The overexpression of MT-1 dramatically reduced the cryolesion-induced oxidative stress and neuronal apoptosis. Remarkably, these effects were also obtained by the intraperitoneal administration of MT-2 to both normal and MT-1+2 knock-out mice. These results fully support the notion that MT-1+2 are essential in the CNS for coping with focal brain injury and suggest a potential therapeutic use of these proteins.

Astrocytic expression of the Alzheimer's disease β-secretase (BACE1) is stimulus-dependent

The beta‐site APP‐cleaving enzyme (BACE1) is a prerequisite for the generation of β‐amyloid peptides, which give rise to cerebrovascular and parenchymal β‐amyloid deposits in the brain of Alzheimers disease patients. BACE1 is neuronally expressed in the brains of humans and experimental animals such as mice and rats. In addition, we have recently shown that BACE1 protein is expressed by reactive astrocytes in close proximity to β‐amyloid plaques in the brains of aged transgenic Tg2576 mice that overexpress human amyloid precursor protein carrying the double mutation K670N‐M671L. To address the question whether astrocytic BACE1 expression is an event specifically triggered by β‐amyloid plaques or whether glial cell activation by other mechanisms also induces BACE1 expression, we used six different experimental strategies to activate brain glial cells acutely or chronically. Brain sections were processed for the expression of BACE1 and glial markers by double immunofluorescence labeling and evaluated by confocal laser scanning microscopy. There was no detectable expression of BACE1 protein by activated microglial cells of the ameboid or ramified phenotype in any of the lesion paradigms studied. In contrast, BACE1 expression by reactive astrocytes was evident in chronic but not in acute models of gliosis. Additionally, we observed BACE1‐immunoreactive astrocytes in proximity to β‐amyloid plaques in the brains of aged Tg2576 mice and Alzheimers disease patients. GLIA 41:169–179, 2003.

Enhanced seizures and hippocampal neurodegeneration following kainic acid‐induced seizures in metallothionein‐I + II‐deficient mice

Metallothioneins (MTs) are major zinc binding proteins in the CNS that could be involved in the control of zinc metabolism as well as in protection against oxidative stress. Mice lacking MT‐I and MT‐II (MT‐I + II deficient) because of targeted gene inactivation were injected with kainic acid (KA), a potent convulsive agent, to examine the neurobiological importance of these MT isoforms. At 35 mg/kg KA, MT‐I + II deficient male mice showed a higher number of convulsions and a longer convulsion time than control mice. Three days later, KA‐injected mice showed gliosis and neuronal injury in the hippocampus. MT‐I + II deficiency decreased both astrogliosis and microgliosis and potentiated neuronal injury and apoptosis as shown by terminal deoxynucleotidyl transferase‐mediated in situ end labelling (TUNEL), detection of single stranded DNA (ssDNA) and by increased interleukin‐1β‐converting enzyme (ICE) and caspase‐3 levels. Histochemically reactive zinc in the hippocampus was increased by KA to a greater extent in MT‐I + II‐deficient compared with control mice. KA‐induced seizures also caused increased oxidative stress, as suggested by the malondialdehyde (MDA) and protein tyrosine nitration (NITT) levels and by the expression of MT‐I + II, nuclear factor‐κB (NF‐κB), and Cu/Zn‐superoxide dismutase (Cu/Zn‐SOD). MT‐I + II deficiency potentiated the oxidative stress caused by KA. Both KA and MT‐I + II deficiency significantly affected the expression of MT‐III, granulocyte‐macrophage colony stimulating factor (GM‐CSF) and its receptor (GM‐CSFr). The present results indicate MT‐I + II as important for neuron survival during KA‐induced seizures, and suggest that both impaired zinc regulation and compromised antioxidant activity contribute to the observed neuropathology of the MT‐I + II‐deficient mice.