Oral glutamine supplementation relieves muscle loss in immobilized rats, altering p38MAPK and FOXO3a signaling pathways.

BACKGROUND: Skeletal muscle synthesizes, stores, and releases body L-glutamine (GLN). Muscle atrophy due to disabling diseases triggers the activation of proteolytic and pro-apoptotic cell signaling, thus impairing the body's capacity to manage GLN content. This situation has a poor therapeutic prognosis. OBJECTIVE: Evaluating if oral GLN supplementation can attenuate muscle wasting mediated by elevated plasma cortisol and activation of caspase-3, p38MAPK, and FOXO3a signaling pathways in soleus and gastrocnemius muscles of rats submitted to 14-day bilateral hindlimbs immobilization. METHODS: Animals were randomly distributed into six groups: non-immobilized rats (Control), control orally supplemented with GLN (1 g kg-1) in solution with L-alanine (ALA: 0.61 g kg-1; GLN+ALA), control orally supplemented with dipeptide L-alanyl-L-glutamine (DIP; 1.49 g kg-1), hindlimbs immobilized rats (IMOB), IMOB orally GLN+ALA supplemented (GLN+ALA-IMOB), and IMOB orally DIP supplemented (DIP-IMOB). Plasma and muscle GLN concentration, plasma cortisol level, muscle caspase-3 activity, muscle p38MAPK and FOXO3a protein content (total and phosphorylated forms), and muscle cross-sectional area (CSA) were measured. RESULTS: Compared to controls, IMOB rats presented: a) increased plasma cortisol levels; b) decreased plasma and muscle GLN concentration; c) increased muscle caspase-3 activity; d) increased total and phosphorylated p38MAPK protein content; e) increased FOXO3a and decreased phosphorylated FOXO3a protein content; f) reduced muscle weight and CSA befitting to atrophy. Oral supplementation with GLN+ALA and DIP was able to significantly attenuate these effects. CONCLUSIONS: These findings attest that oral GLN supplementation in GLN+ALA solution or DIP forms attenuates rats' skeletal muscle mass wasting caused by disuse-mediated muscle atrophy.

Functional Recovery Caused by Human Adipose Tissue Mesenchymal Stem Cell-Derived Extracellular Vesicles Administered 24 h after Stroke in Rats.

Ischemic stroke is a major cause of death and disability, intensely demanding innovative and accessible therapeutic strategies. Approaches presenting a prolonged period for therapeutic intervention and new treatment administration routes are promising tools for stroke treatment. Here, we evaluated the potential neuroprotective properties of nasally administered human adipose tissue mesenchymal stem cell (hAT-MSC)-derived extracellular vesicles (EVs) obtained from healthy individuals who underwent liposuction. After a single intranasal EV (200 µg/kg) administered 24 h after a focal permanent ischemic stroke in rats, a higher number of EVs, improvement of the blood-brain barrier, and re-stabilization of vascularization were observed in the recoverable peri-infarct zone, as well as a significant decrease in infarct volume. In addition, EV treatment recovered long-term motor (front paws symmetry) and behavioral impairment (short- and long-term memory and anxiety-like behavior) induced by ischemic stroke. In line with these findings, our work highlights hAT-MSC-derived EVs as a promising therapeutic strategy for stroke.

Resveratrol increases the activation markers and changes the release of inflammatory cytokines of hepatic stellate cells.

The phytoalexin Resveratrol (3,5,4'-trihydroxystilbene; RSV) has been related to numerous beneficial effects on health by its cytoprotection and chemoprevention activities. Liver fibrosis is characterized by the extracellular matrix accumulation after hepatic injury and can lead to cirrhosis. Hepatic stellate cells (HSC) play a crucial role during fibrogenesis and liver wound healing by changing their quiescent phenotype to an activated phenotype for protecting healthy areas from damaged areas. Strategies on promoting the activated HSC death, the quiescence return or the cellular activation stimuli decrease play an important role on reducing liver fibrosis. Here, we evaluated the RSV effects on some markers of activation in GRX, an HSC model. We further evaluated the RSV influence in the ability of GRX on releasing inflammatory mediators. RSV at 1 and 10 µM did not alter the protein content of α-SMA, collagen I and GFAP; but 50 µM increased the content of these activation-related proteins. Also, RSV did not change the myofibroblast-like morphology of GRX. Interestingly, RSV at 10 and 50 µM decreased the GRX migration and collagen-I gel contraction. Finally, we showed that RSV triggered the increase in the TNF-α and IL-10 content in culture media of GRX while the opposite occurred for the IL-6 content. Altogether, these results suggested that RSV did not decrease the activation state of GRX and oppositely, triggered a pro-activation effect at the 50 µM concentration. However, despite the increase of TNF- α in culture media, these results on IL-6 and IL-10 secretion were in accordance with the anti-inflammatory role of RSV in our model.

TRF1 as a major contributor for telomeres' shortening in the context of obesity.

Obesity is a prevalent multifactorial chronic disorder characterized by metabolic dysregulation. Sustained pro-oxidative mediators trigger harmful consequences that reflect at systemic level and contribute for the establishment of a premature senescent phenotype associated with macromolecular damage (DNA, protein, and lipids). Telomeres are structures that protect chromosome ends and are associated with a six-protein complex called the shelterin complex and subject to regulation. Under pro-oxidant conditions, telomere attrition and the altered expression of the shelterin proteins are central for the establishment of many pathophysiological conditions such as obesity. Thus, considering that individuals with obesity display a systemic oxidative stress profile that may compromise the telomeres length or its regulation, the aim of this study was to investigate telomere homeostasis in patients with obesity and explore broad/systemic associations with the expression of shelterin genes and the plasma redox state. We performed a cross-sectional study in 39 patients with obesity and 27 eutrophic subjects. Telomere length (T/S ratio) and gene expression of shelterin components were performed in peripheral blood mononuclear cells by qPCR. The oxidative damage (lipid peroxidation and protein carbonylation) and non-enzymatic antioxidant system (total radical-trapping antioxidant potential/reactivity, sulfhydryl and GSH content) were evaluated in plasma. Our results demonstrate that independently of comorbidities, individuals with obesity had significantly shorter telomeres, augmented expression of negative regulators of the shelterin complex, increased lipid peroxidation and higher oxidized protein levels associated with increased non-enzymatic antioxidant defenses. Principal component analysis revealed TRF1 as a major contributor for firstly telomeres shortening. In conclusion, our study is first showing a comprehensive analysis of telomeres in the context of obesity, associated with dysregulation of the shelterin components that was partially explained by TRF1 upregulation that could not be reversed by the observed adaptive non-enzymatic antioxidant response.

Immunosenescence Induced by Plasma from Individuals with Obesity Caused Cell Signaling Dysfunction and Inflammation.

OBJECTIVE: To evaluate the consequences of plasma from individuals with obesity on parameters associated with immunosenescence in unrelated healthy peripheral blood mononuclear cells (PBMC). METHODS: Freshly isolated PBMC were incubated in media supplemented with 10% of plasma from individuals with obesity or control subjects for the first 4 hours of 24 to 120 hours of culture. RESULTS: Plasma from individuals with obesity modulated the phenotype of healthy PBMC, leading to a higher rate of apoptosis, lower amounts of phospho-γH2AX and -p53, and mitochondrial dysfunction. After 120 hours, there was a higher secretion of inflammatory cytokines IL-1ß and IL-8. CD8+ T lymphocytes presented decreased expression of CD28, which is associated with the immunosenescent phenotype. CD14+ macrophages showed increased expression of CD80 and CD206, suggesting a modulation in the activation of macrophages. CONCLUSIONS: These results demonstrate that chronic systemic inflammation observed in obesity induces dysfunctional features in PBMC that are consistent with premature immunosenescence.

Hypoxic and Reoxygenated Microenvironment: Stemness and Differentiation State in Glioblastoma.

Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor in adults. Hypoxia is a distinct feature in GBM and plays a significant role in tumor progression, resistance to treatment, and poor outcome. However, there is lack of studies relating type of cell death, status of Akt phosphorylation on Ser473, mitochondrial membrane potential, and morphological changes of tumor cells after hypoxia and reoxygenation. The rat glioma C6 cell line was exposed to oxygen deprivation (OD) in 5 % fetal bovine serum (FBS) or serum-free media followed by reoxygenation (RO). OD induced apoptosis on both 5 % FBS and serum-free groups. Overall, cells on serum-free media showed more profound morphological changes than cells on 5 % FBS. Moreover, our results suggest that OD combined with absence of serum provided a favorable environment for glioblastoma dedifferentiation to cancer stem cells, since nestin, and CD133 levels increased. Reoxygenation is present in hypoxic tumors through microvessel formation and cell migration to oxygenated areas. However, few studies approach these phenomena when analyzing hypoxia. We show that RO caused morphological alterations characteristic of cells undergoing a differentiation process due to increased GFAP. In the present study, we characterized an in vitro hypoxic microenvironment associated with GBM tumors, therefore contributing with new insights for the development of therapeutics for resistant glioblastoma.

Phosphatidylinositol 3-Kinase/AKT Pathway Inhibition by Doxazosin Promotes Glioblastoma Cells Death, Upregulation of p53 and Triggers Low Neurotoxicity.

Glioblastoma is the most frequent and malignant brain tumor. Treatment includes chemotherapy with temozolomide concomitant with surgical resection and/or irradiation. However, a number of cases are resistant to temozolomide, as well as the human glioblastoma cell line U138-MG. We investigated doxazosin's (an antihypertensive drug) activity against glioblastoma cells (C6 and U138-MG) and its neurotoxicity on primary astrocytes and organoptypic hippocampal cultures. For this study, the following methods were used: citotoxicity assays, flow cytometry, western-blotting and confocal microscopy. We showed that doxazosin induces cell death on C6 and U138-MG cells. We observed that doxazosin's effects on the PI3K/Akt pathway were similar as LY294002 (PI3K specific inhibitor). In glioblastoma cells treated with doxasozin, Akt levels were greatly reduced. Upon examination of activities of proteins downstream of Akt we observed upregulation of GSK-3ß and p53. This led to cell proliferation inhibition, cell death induction via caspase-3 activation and cell cycle arrest at G0/G1 phase in glioblastoma cells. We used in this study Lapatinib, a tyrosine kinase inhibitor, as a comparison with doxazosin because they present similar chemical structure. We also tested the neurocitotoxicity of doxazosin in primary astrocytes and organotypic cultures and observed that doxazosin induced cell death on a small percentage of non-tumor cells. Aggressiveness of glioblastoma tumors and dismal prognosis require development of new treatment agents. This includes less toxic drugs, more selective towards tumor cells, causing less damage to the patient. Therefore, our results confirm the potential of doxazosin as an attractive therapeutic antiglioma agent.

Resveratrol inhibits cell growth by inducing cell cycle arrest in activated hepatic stellate cells.

Resveratrol (RSV) exerts anti-proliferative and pro-apoptotic actions in different cell lines. Hepatic stellate cells (HSCs) are major fibrogenic cell types that contribute to collagen accumulation during chronic liver disease. In the present study, the inhibitory effects of RSV on cell proliferation, cell cycle, and apoptosis were evaluated in the mouse hepatic stellate cell line GRX. Cells treated with 1 nM-1 muM of RSV demonstrated a decrease in cell growth of about 35% after 5 days. GRX cells, treated with RSV (100 nM or 1 muM), were analyzed by flow cytometry; RSV induced an increase in the number of GRX cells in the S- and sub-G1 phases. The increase in sub-G1 phase cells and the nuclear condensation and fragmentation shown by DAPI staining identified a possible pro-apoptotic effect of RSV on GRX cells. Furthermore, the RSV anti-proliferative effects could be explained by an S-phase accumulation caused by a decrease in the progression through the cell cycle or an inhibition of S or G2 phase transition. It is notable that these RSV actions are mediated at nanomolar levels, compatible with the concentrations of free RSV in biological fluids after ingestion of polyphenol-rich foods, suggesting a possible effect of these foods as an adjuvant treatment in chronic liver diseases.

Hyperthyroidism in the developing rat testis is associated with oxidative stress and hyperphosphorylated vimentin accumulation.

Hyperthyroidism was induced in rats and somatic indices and metabolic parameters were analyzed in testis. In addition, the morphological analysis evidenced testes maturation and intense protein synthesis and processing, supporting the enhancement in vimentin synthesis in hyperthyroid testis. Furthermore, vimentin phosphorylation was increased, indicating an accumulation of phosphorylated vimentin associated to the cytoskeleton, which could be a consequence of the extracellular-regulated kinase (ERK) activation regulating the cytoskeleton. Biomarkers of oxidative stress demonstrated an increased basal metabolic rate measured by tissue oxygen consumption, as well as, increased TBARS levels. In addition, the enzymatic and non-enzymatic antioxidant defences appeared to respond according to the augmented oxygen consumption. We observed decreased total glutathione levels, with enhancement of reduced glutathione, whereas most of the antioxidant enzyme activities were induced. Otherwise, superoxide dismutase activity was inhibited. These results support the idea that an increase in mitochondrial ROS generation, underlying cellular oxidative damage, is a side effect of hyperthyroid-induced biochemical changes by which rat testis increase their metabolic capacity.