PGC-1α activation boosts exercise-dependent cellular response in the skeletal muscle.

The role of Peroxisome proliferator-activated receptor-gamma coactivator alpha (PGC-1α) in fat metabolism is not well known. In this study, we compared the mechanisms of muscle-specific PGC-1α overexpression and exercise-related adaptation-dependent fat metabolism. PGC-1α trained (PGC-1α Ex) and wild-trained (wt-ex) mice were trained for 10 weeks, five times a week at 30 min per day with 60 percent of their maximal running capacity. The PGC-1α overexpressed animals exhibited higher levels of Fibronectin type III domain-containing protein 5 (FNDC5), 5' adenosine monophosphate-activated protein kinase alpha (AMPK-α), the mammalian target of rapamycin (mTOR), Sirtuin 1 (SIRT1), Lon protease homolog 1 (LONP1), citrate synthase (CS), succinate dehydrogenase complex flavoprotein subunit A (SDHA), Mitofusin-1 (Mfn1), endothelial nitric oxide synthase (eNOS), Hormone-sensitive lipase (HSL), adipose triglyceride lipase (ATGL), G protein-coupled receptor 41 (GPR41), and Phosphatidylcholine Cytidylyltransferase 2 (PCYT2), and lower levels of Sirtuin 3 (SIRT3) compared to wild-type animals. Exercise training increased the protein content levels of SIRT1, HSL, and ATGL in both the wt-ex and PGC-1α trained groups. PGC-1α has a complex role in cellular signaling, including the upregulation of lipid metabolism-associated proteins. Our data reveals that although exercise training mimics the effects of PGC-1α overexpression, it incorporates some PGC-1α-independent adaptive mechanisms in fat uptake and cell signaling.

Exercise combined with postbiotics treatment results in synergistic improvement of mitochondrial function in the brain of male transgenic mice for Alzheimer's disease.

BACKGROUND: It has been suggested that exercise training and postbiotic supplement could decelerate the progress of functional and biochemical deterioration in double transgenic mice overexpresses mutated forms of the genes for human amyloid precursor protein (APPsw) and presenilin 1 (m146L) (APP/PS1TG). Our earlier published data indicated that the mice performed better than controls on the Morris Maze Test parallel with decreased occurrence of amyloid-ß plaques in the hippocampus. We investigated the neuroprotective and therapeutic effects of high-intensity training and postbiotic supplementation. METHODS: Thirty-two adult APP/PS1TG mice were randomly divided into four groups: (1) control, (2) high-intensity training (3) postbiotic, (4) combined (training and postbiotic) treatment for 20 weeks. In this study, the whole hemibrain without hippocampus was used to find molecular traits explaining improved brain function. We applied qualitative RT-PCR for gene expression, Western blot for protein level, and Zymography for LONP1 activity. Disaggregation analysis of Aß-40 was performed in the presence of Lactobacillus acidophilus and Bifidobacterium longum lysate. RESULTS: We found that exercise training decreased Alzheimer's Disease (AD)-related gene expression (NF-kB) that was not affected by postbiotic treatment. The preparation used for postbiotic treatment is composed of tyndallized Bifidobacterium longum and Lactobacillus acidophilus. Both of the postbiotics effectively disaggregated amyloid-ß/Aß-40 aggregates by chelating Zn2+ and Cu2+ ions. The postbiotic treatment decreased endogenous human APPTG protein expression and mouse APP gene expression in the hemibrains. In addition, the postbiotic treatment elevated mitochondrial LONP1 activity as well. CONCLUSION: Our findings revealed distinct mechanisms behind improved memory performance in the whole brain: while exercise training modulates NF-kB signaling pathway regulating immune response until postbiotic diminishes APP gene expression, disaggregates pre-existing amyloid-ß plaques and activates mitochondrial protein quality control in the region of brain out of hippocampus. Using the above treatments complements and efficiently slows down the development of AD.

DNA methylation clock DNAmFitAge shows regular exercise is associated with slower aging and systemic adaptation.

DNAmPhenoAge, DNAmGrimAge, and the newly developed DNAmFitAge are DNA methylation (DNAm)-based biomarkers that reflect the individual aging process. Here, we examine the relationship between physical fitness and DNAm-based biomarkers in adults aged 33-88 with a wide range of physical fitness (including athletes with long-term training history). Higher levels of VO2max (ρ = 0.2, p = 6.4E - 4, r = 0.19, p = 1.2E - 3), Jumpmax (p = 0.11, p = 5.5E - 2, r = 0.13, p = 2.8E - 2), Gripmax (ρ = 0.17, p = 3.5E - 3, r = 0.16, p = 5.6E - 3), and HDL levels (ρ = 0.18, p = 1.95E - 3, r = 0.19, p = 1.1E - 3) are associated with better verbal short-term memory. In addition, verbal short-term memory is associated with decelerated aging assessed with the new DNAm biomarker FitAgeAcceleration (ρ: - 0.18, p = 0.0017). DNAmFitAge can distinguish high-fitness individuals from low/medium-fitness individuals better than existing DNAm biomarkers and estimates a younger biological age in the high-fit males and females (1.5 and 2.0 years younger, respectively). Our research shows that regular physical exercise contributes to observable physiological and methylation differences which are beneficial to the aging process. DNAmFitAge has now emerged as a new biological marker of quality of life.

Master athletes have higher miR-7, SIRT3 and SOD2 expression in skeletal muscle than age-matched sedentary controls.

Regular physical exercise has health benefits and can prevent some of the ageing-associated muscle deteriorations. However, the biochemical mechanisms underlying this exercise benefit, especially in human tissues, are not well known. To investigate, we assessed this using miRNA profiling, mRNA and protein levels of anti-oxidant and metabolic proteins in the vastus lateralis muscle of master athletes aged over 65 years and age-matched controls. Master athletes had lower levels of miR-7, while mRNA or protein levels of SIRT3, SIRT1, SOD2, and FOXO1 levels were significantly higher in the vastus lateralis muscle of master athletes compared to muscles of age-matched controls. These results suggest that regular exercise results in better cellular metabolism and antioxidant capacity via maintaining physiological state of mitochondria and efficient ATP production and decreasing ageing-related inflammation as indicated by the lower level of miR-7 in master athletes.

SIRT1 may play a crucial role in overload-induced hypertrophy of skeletal muscle.

KEY POINTS: Silent mating type information regulation 2 homologue 1 (SIRT1) activity and content increased significantly in overload-induced hypertrophy. SIRT1-mediated signalling through Akt, the endothelial nitric oxide synthase mediated pathway, regulates anabolic process in the hypertrophy of skeletal muscle. The regulation of catabolic signalling via forkhead box O 1 and protein ubiquitination is SIRT1 dependent. Overload-induced changes in microRNA levels regulate SIRT1 and insulin-like growth factor 1 signalling. ABSTRACT: Significant skeletal muscle mass guarantees functional wellbeing and is important for high level performance in many sports. Although the molecular mechanism for skeletal muscle hypertrophy has been well studied, it still is not completely understood. In the present study, we used a functional overload model to induce plantaris muscle hypertrophy by surgically removing the soleus and gastrocnemius muscles in rats. Two weeks of muscle ablation resulted in a 40% increase in muscle mass, which was associated with a significant increase in silent mating type information regulation 2 homologue 1 (SIRT1) content and activity (P < 0.001). SIRT1-regulated Akt, endothelial nitric oxide synthase and GLUT4 levels were also induced in hypertrophied muscles, and SIRT1 levels correlated with muscle mass, paired box protein 7 (Pax7), proliferating cell nuclear antigen (PCNA) and nicotinamide phosphoribosyltransferase (Nampt) levels. Alternatively, decreased forkhead box O 1 (FOXO1) and increased K48 polyubiquitination also suggest that SIRT1 could be involved in the catabolic process of hypertrophy. Furthermore, increased levels of K63 and muscle RING finger 2 (MuRF2) protein could also be important enhancers of muscle mass. We report here that the levels of miR1 and miR133a decrease in hypertrophy and negatively correlate with muscle mass, SIRT1 and Nampt levels. Our results reveal a strong correlation between SIRT1 levels and activity, SIRT1-regulated pathways and overload-induced hypertrophy. These findings, along with the well-known regulatory roles that SIRT1 plays in modulating both anabolic and catabolic pathways, allow us to propose the hypothesis that SIRT1 may actually play a crucial causal role in overload-induced hypertrophy of skeletal muscle. This hypothesis will now require rigorous direct and functional testing.

Eating habits modulate short term memory and epigenetical regulation of brain derived neurotrophic factor in hippocampus of low- and high running capacity rats.

Exercise capacity and dietary restriction (DR) are linked to improved quality of life, including enhanced brain function and neuro-protection. Brain derived neurotrophic factor (BDNF) is one of the key proteins involved in the beneficial effects of exercise on brain. Low capacity runner (LCR) and high capacity runner (HCR) rats were subjected to DR in order to investigate the regulation of BDNF. HCR-DR rats out-performed other groups in a passive avoidance test. BDNF content increased significantly in the hippocampus of HCR-DR groups compared to control groups (p<0.05). The acetylation of H3 increased significantly only in the LCR-DR group. However, chip-assay revealed that the specific binding between acetylated histone H3 and BNDF promoter was increased in both LCR-DR and HCR-DR groups. In spite of these increases in binding, at the transcriptional level only, the LCR-DR group showed an increase in BDNF mRNA content. Additionally, DR also induced the activity of cAMP response element-binding protein (CREB), while the content of SIRT1 was not altered. Peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) was elevated in HCR-DR groups. But, based on the levels of nuclear respiratory factor-1 and cytocrome c oxidase, it appears that DR did not cause mitochondrial biogenesis. The data suggest that DR-mediated induction of BDNF levels includes chromatin remodeling. Moreover, DR does not induce mitochondrial biogenesis in the hippocampus of LCR/HCR rats. DR results in different responses to a passive avoidance test, and BDNF regulation in LCR and HCR rats.

High altitude exposure alters gene expression levels of DNA repair enzymes, and modulates fatty acid metabolism by SIRT4 induction in human skeletal muscle.

We hypothesized that high altitude exposure and physical activity associated with the attack to Mt Everest could alter mRNA levels of DNA repair and metabolic enzymes and cause oxidative stress-related challenges in human skeletal muscle. Therefore, we have tested eight male mountaineers (25-40 years old) before and after five weeks of exposure to high altitude, which included attacks to peaks above 8000m. Data gained from biopsy samples from vastus lateralis revealed increased mRNA levels of both cytosolic and mitochondrial superoxide dismutase. On the other hand 8-oxoguanine DNA glycosylase (OGG1) mRNA levels tended to decrease while Ku70 mRNA levels and SIRT6 decreased with altitude exposure. The levels of SIRT1 and SIRT3 mRNA did not change significantly. However, SIRT4 mRNA level increased significantly, which could indicate decreases in fatty acid metabolism, since SIRT4 is one of the important regulators of this process. Within the limitations of this human study, data suggest that combined effects of high altitude exposure and physical activity climbing to Mt. Everest, could jeopardize the integrity of the particular chromosome.

Influence of pulsing electromagnetic field therapy on resting blood pressure in aging adults.

This double-blind study tested the effects of pulsating electromagnetic field (PEMF) therapy sessions on the changes in peripheral cardiovascular function in a group of aging adults after 12 weeks of treatment. Each therapy session involved 15 min of exposure to low-frequency PEMF with asymmetrical waveforms emitted by the Impulser™ Pro mattress. The treatment was provided 5 days per week for a total of 60 sessions. Resting blood pressure and arterial stiffness index were determined for peripheral cardiovascular function. Fifty-four older men and women (mean age 59.8 ± 3.5 yrs) completed the entire protocol involving either the PEMF or a sham treatment. The results include statistically significant reductions in systolic and pulse blood pressure, while no significant difference in diastolic pressure or the index of arterial stiffness was observed. These findings suggest that the PEMF treatment might be linked to improvements in peripheral resistance or circulation.

The effects of aging, physical training, and a single bout of exercise on mitochondrial protein expression in human skeletal muscle.

Aging results in a significant decline in aerobic capacity and impaired mitochondrial function. We have tested the effects of moderate physical activity on aerobic capacity and a single bout of exercise on the expression profile of mitochondrial biogenesis, and fusion and fission related genes in skeletal muscle of human subjects. Physical activity attenuated the aging-associated decline in VO2 max (p<0.05). Aging increased and a single exercise bout decreased the expression of nuclear respiratory factor-1 (NRF1), while the transcription factor A (TFAM) expression showed a strong relationship with VO(2max) and increased significantly in the young physically active group. Mitochondrial fission representing FIS1 was induced by regular physical activity, while a bout of exercise decreased fusion-associated gene expression. The expression of polynucleotide phosphorylase (PNPase) changed inversely in young and old groups and decreased with aging. The A2 subunit of cyclic AMP-activated protein kinase (AMPK) was induced by a single bout of exercise in skeletal muscle samples of both young and old subjects (p<0.05). Our data suggest that moderate levels of regular physical activity increases a larger number of mitochondrial biogenesis-related gene expressions in young individuals than in aged subjects. Mitochondrial fission is impaired by aging and could be one of the most sensitive markers of the age-associated decline in the adaptive response to physical activity.

Age-dependent changes in 8-oxoguanine-DNA glycosylase activity are modulated by adaptive responses to physical exercise in human skeletal muscle.

8-Oxo-7,8-dihydroguanine (8-oxoG) accumulates in the genome over time and is believed to contribute to the development of aging characteristics of skeletal muscle and various aging-related diseases. Here, we show a significantly increased level of intrahelical 8-oxoG and 8-oxoguanine-DNA glycosylase (OGG1) expression in aged human skeletal muscle compared to that of young individuals. In response to exercise, the 8-oxoG level was lastingly elevated in sedentary young and old subjects, but returned rapidly to preexercise levels in the DNA of physically active individuals independent of age. 8-OxoG levels in DNA were inversely correlated with the abundance of acetylated OGG1 (Ac-OGG1), but not with total OGG1, apurinic/apyrimidinic endonuclease 1 (APE1), or Ac-APE1. The actual Ac-OGG1 level was linked to exercise-induced oxidative stress, as shown by changes in lipid peroxide levels and expression of Cu,Zn-SOD, Mn-SOD, and SIRT3, as well as the balance between acetyltransferase p300/CBP and deacetylase SIRT1, but not SIRT6 expression. Together these data suggest that that acetylated form of OGG1, and not OGG1 itself, correlates inversely with the 8-oxoG level in the DNA of human skeletal muscle, and the Ac-OGG1 level is dependent on adaptive cellular responses to physical activity, but is age independent.

Preliminary studies of the effects of vascular adhesion protein-1 inhibitors on experimental corneal neovascularization.

Vascular adhesion protein-1 (VAP-1) controls the adhesion of lymphocytes to endothelial cells and is upregulated at sites of inflammation. Moreover, it expresses amine oxidase activity, due to the sequence identity with semicarbazide-sensitive amine oxidase. Recent studies indicate a significant role for VAP-1 in neovascularization, besides its contribution to inflammation. Pathological blood vessel development in severe ocular diseases (such as diabetes, age-related macula degeneration, trauma and infections) might lead to decreased visual acuity and finally to blindness, yet there is no clear consensus as to its appropriate treatment. In the present case study, the effects of two VAP-1 inhibitors on experimentally induced corneal neovascularization in rabbits were compared with the effects of a known inhibitor of angiogenesis, bevacizumab, an anti-vascular endothelial growth factor antibody. In accordance with recent literature data, the results of the preliminary study reported here indicate that the administration of VAP-1 inhibitors is a potentially valuable therapeutic option in the treatment of corneal neovascularization.

Reverse regulation of endothelial cells and myointimal hyperplasia on cell proliferation by a heatshock protein-coinducer after hypoxia.

BACKGROUND AND PURPOSE: Myointimal hyperplasia (MIH) cells are related to permanent upregulated proliferation as tumor-like cells. The aim of this study is to assess whether treatment of cells after hypoxia by Iroxanadine heat-shock protein (HSP-coinducer) predicts recovery through cell proliferation. METHODS: Vascular smooth muscle cells (VSMC) and brain capillary endothelial cells (HBEC) were isolated from human origin and MIH-cells from early carotid restenosis after surgery. Cell proliferation was quantified by bromuridine (BrdU) incorporation after hypoxia/reoxygenation. HSP72 and cyclin-dependent kinase (CDKN1A) mRNA expression was assessed by reverse transcription-polymerase chain reaction (PCR) and cell cycle distribution by flow cytometry (FACS) analysis. RESULTS: After hypoxia/reoxygenation, the proliferation of MIH-cells increased, whereas endothelial cells decreased (MIH: 0.266+/-0.016 versus 0.336+/-0.024; P<0.05; HBEC: 1.249+/-0.10 versus 0.878+/-0.11; P<0.05). Whereas augmented proliferation of MIH-cells was reduced (40% to 45%) by HSP-coinducer, diminished HBEC proliferation increased (46.2%). Stress-activated-protein-kinase (SAPK)p38-dependent cell cycle redistribution was generated by an increase in HSP72 and CDKN1A mRNA levels in MIH-cells. CONCLUSIONS: The 2 key players of early restenosis (MIH, EC) were oppositely regulated and correspondingly after treatment by HSP-coinducer reverse recovered. Drug candidate may have therapeutic potential in (re)restenosis.

Cytoprotective effect of two synthetic enhancer substances, (-)-BPAP and (-)-deprenyl, on human brain capillary endothelial cells and rat PC12 cells.

Enhancer regulation is a new control mechanism in the brain [Knoll, J., 2003. Enhancer regulation/endogenous and synthetic enhancer compounds: a neurochemical concept of the innate and acquired drives. Neurochemical Research 28(8), 1275-1297]. Enhancer substances exert their effect in bi-modal form with a highly characteristic dose-dependency. Two bell-shaped concentration curves have been published. The one in ultra low concentration range (fM) specific form of enhancer regulation and the other at high concentration (100 microM) non-specific form of enhancer regulation. Catecholaminergic neurons proved to be enhancer-sensitive cells. Since rat PC12 cells and human brain endothelial cells (HBEC) work under catecholaminergic influence, it was reasonable to expect that both the specific and non-specific form of the enhancer regulation might be detectable in these cells. We tested this possibility on these cultured cells under normoxia and hypoxia-reoxygenation. After 1 h hypoxia produced by Argon gas and 0, 2, 4, and 20 h reoxygenation the cell loss was calculated by propidiumiodide assay and the cell activity was investigated by Alamar Blue assay colorimetric measurement. The percentages of living and necrotic cells were expressed after propidiumiodide staining. Broad scale concentrations of the two compounds (1 fM-100 microM) were added to the culture strait after the oxygen deprivation. (-)-BPAP and (-)-deprenyl, due to their enhancer effect, exerted a significant cytoprotective effect on both HBECs and PC12 cells. In harmony with Knoll's publications we were able to demonstrate by the aid of (-)-BPAP and (-)-deprenyl that both HBEC and PC12 are enhancer-sensitive cells. We detected the specific form of the enhancer regulation in the ultra low concentration range (fM-pM) and also the non-specific form of the enhancer regulation was visible (mM-microM).

Human cerebral microvessel endothelial cell culture as a model system to study the blood-brain interface in ischemic/hypoxic conditions.

(1) Cerebral ischemia and reperfusion induce several changes on the endothelial cells at the microcirculatory level. (2) Vasogenic brain edema due to compromised blood-brain barrier, transformation of the endothelial cell surface from an anticoagulant to a procoagulant surface are important factors in the pathogenesis of ischemic stroke. (3) Release of prostaglandins, endothelin-1, complement proteins, and matrix metalloproteinase-9 by microvascular endothelial cells are other components in the complex mechanism of brain ischemia/hypoxia. (4) Ultrastructural studies documented the opened paracellular avenues in the course of vasogenic edema in different experimental models (5) Tight junctions of endothelial cells have been characterized with freeze fracture electron microscopy, and the process of transvesiculation was analyzed using rapid freeze and freeze substitution procedure before electron microscopy studies (6) In endothelial cell-culture experiments, we used rodent and later human brains. (7) Endothelial cells co-cultured with astroglia resulted in an elaborate tight junctional complex. (8) This co-culture technique becomes the basis of in vitro blood-brain barrier studies On endothelial cells of human brain origin, different regulatory factors found to be responsible for the complex mechanism of ischemic stroke.

[Regulatory mechanisms in focal cerebral ischemia. New possibilities in neuroprotective therapy]. / Szabályozó mechanizmusok a fokális agyi ischaemia kialakulásában. A neuroprotektív kezeles uj lehetóségei.

Permanent or temporary disruption of cerebral blood flow rapidly depletes brain regions of their limited energy reserves (glycogen, glucose, oxygen, ATP) leading to an energy crisis. Tissue damage occurs due to the energy crisis. The central part of the damage, the ischaemic "core" region is surrounded by zones of the shell-like penumbra. Necrotic, as well as apoptotic cell death could be identified in the penumbra. Going away from the ischaemic core different neurochemical processes are occurring by space and time. "Immediate early response" genes (c-fos, fos-B, c-Jun, krox 20, 24) are activated, heatshock proteins (hsp 70, 72, HSF, HSE, HIF), cytokines (TNF-alpha, IL-1 beta), inflammatory factors (COX), adhesion and glial factors (ICAM-1, ELAM-1, P-selectin), vasoactive factors (IL-6, -10, PAF), reactive oxigen radicals and connected factors (O2, OH, NO, NOS, SOD) are produced within minutes and hours. Cell deaths, necrosis and apoptosis due to the activation of calpains, caspases and nucleases occur in days. In parallel, growth factors and plasticity proteins (BDNF, NGF, TGF-beta, VEGF, PDGF, GAP-43) are activated as a basis of functional rehabilitation.

Effect of a single high-dose gamma irradiation on cultured cells in human cerebral arteriovenous malformation.

OBJECT: The purpose of this study was to analyze the effect of single high-dose gamma irradiation at a cellular biological level on tissue cultures obtained in patients who underwent surgery for cerebral arteriovenous malformation (AVM). METHODS: The cell proliferation indices and changes in activation of p53, p21Waf-1, and mdm-2 were determined. Additionally, immunohistochemical investigations for vimentin, desmin, alpha-smooth muscle actin (alpha-SMA), glial fibrillary acidic protein, Factor VIII-related antigen (F-VIII), cytokeratin, S100, and transforming growth factor-beta (TGFbeta) were performed on cultured AVM cells after a single high-dose irradiation. Normal human brain microvessel endothelial (HBE) cells and aortic smooth muscle cells served as controls. The proliferation index decreased on the 5th day after irradiation and remained depressed over the observation period in the irradiated AVM cultures. The p53, p21Waf-1, and mdm-2 messenger RNA measurements showed considerable elevation both in AVM cultures and HBE cells after 15-Gy irradiation, which indicated apoptosis. Immunohistochemistry revealed strong vimentin positivity in the nonirradiated cultures, which gradually decreased in the irradiated cultures. Transforming growth factor-beta positivity was demonstrated in the irradiated specimens, indicating transformation of fibroblastic cells into activated myofibroblastic elements. This transformation was confirmed by demonstrating elevated SMA expression as well in the radiation-treated fibroblasts. CONCLUSIONS: The presence of TGFbeta and alpha-SMA activity in the irradiated AVM cells suggests that along with the genetically confirmed apoptotic activity, fibroblast transformation into myofibroblasts might be one of the mechanisms leading to shrinkage and obliteration of AVMs after single high-dose gamma irradiation.