Search

Methotrexate (MTX) is broadly used in the treatment of chronic inflammatory diseases such as rheumatoid arthritis (RA). The prevalence of metabolic syndrome (MeS) in patients with this condition is relatively high. Given the importance of adipose tissue in the development of obesity metabolic complications, this study aimed to investigate the effect of methotrexate on preadipocyte proliferation, adipogenesis, and glucose uptake by adipocytes. 3T3-L1 preadipocytes proliferation was evaluated by sulforhodamine B staining and H-3-thymidine incorporation, after 24 or 48 h of treatment with MTX (0.1 and 10 mu M). Preadipocytes were induced to differentiate with an appropriate adipogenic cocktail in the presence or absence of MTX. Adipogenesis was determined by measuring lipid accumulation after staining with oil red O. H-3-Deoxyglucose (H-3-DG) uptake was determined by liquid scintillation counting. MTX treatment reduced culture protein content in a concentration-dependent manner and H-3-thymidine incorporation (P < 0.05). MTX (0.1 mu M) treatment increased lipid accumulation and basal H-3-DG uptake by adipocytes (P < 0.05). In 0.1 mu M MTX-treated adipocytes, insulin stimulation did not result in an increase of H-3-DG uptake, contrarily to what was observed in control cells. These results demonstrate that methotrexate interferes with adipocyte proliferation and promotes the hypertrophic growth of adipocytes. These molecular effects may have implications on metabolic profile of RA patients treated with MTX.

The aim of the present study was to analyze the impact of Loughborough Intermittent Shuttle Test (LIST) versus soccer match on heart rate (HR), muscle damage, redox status, blood leukocytes and neuromuscular function throughout 72 h recovery. Sixteen male soccer players (21.3 +/- A 1.1 years; 175.0 +/- A 6.0 cm; 70.7 +/- A 6.3 kg) completed LIST and performed a soccer match separated by 2 weeks and data were collected before, 30 min, 24, 48 and 72 h after LIST and match. HR, plasma creatine kinase (CK) activity, myoglobin (Mb), uric acid (UA), protein sulfhydryls (-SH), malondialdehyde (MDA) contents, total antioxidant status (TAS), blood leukocyte counts, delayed onset muscle soreness, 20 m sprint and jump performances, and maximal isokinetic knee extension and flexion were analyzed. HR after LIST was significantly lower than after the match. Post-match TAS was lower and UA was higher than after LIST. Thirty minutes and 24 h after soccer MDA was higher and -SH was lower than after LIST (P < 0.05). LIST and soccer match induced elevation in total leukocytes and a reduction in lymphocytes at 30 min. This reduction in blood lymphocytes 30 min after match was lower than after LIST. In conclusion, the impact of both exercises did not differ regarding the observed muscle damage markers and some neuromuscular parameters, although soccer requires higher cardiac demand and induced higher changes on redox status, adenine nucleotide metabolism and on lymphocyte counts than LIST, which should be taken into account when using LIST to simulate a match to study these type of physiological and biochemical-related endpoints.

The purpose of the present study was to investigate the mechanisms of cell death (apoptosis vs. necrosis) during muscle atrophy induced by 1 week of hindlimb suspension. Biochemical and morphological parameters were examined in murine soleus and gastrocnemius muscles. A total of 70 male Charles River CD1 mice were randomly assigned to seven groups (n = 10/group): Cont (loading control conditions) and 6HS, 12HS, 24HS, 48HS, 72HS and 1wkHS with respect to the period of hindlimb suspension (HS). Compared to the Cont, skeletal muscle atrophy was confirmed by a significant decrease of 44 and of 17% in fiber cross-sectional areas in the gastrocnemius and soleus, respectively. A significant increase in caspase-3 activity was noticed in 6HS (196%, P < 0.05) and in 12HS (201%, P < 0.05), as well as the amount of cytosolic mono- and oligonucleosomes at 12HS (142%, P < 0.05) and 24HS (203%, P < 0.05) in the gastrocnemius and soleus, respectively. The profile of necrotic markers showed a peak of myeloperoxidase activity at 24HS (170%, P < 0.05) and at 72HS (114%, P < 0.05) in the gastrocnemius and soleus, respectively. The analysis of N-acetylglucosaminidase activity evidenced more increment in the soleus at 72HS (60%, P < 0.05). The analysis of the basal values of these parameters suggested that apoptosis prevailed in the slow-twitch muscle analyzed, whereas lysosomic activity seemed to be more pronounced in the gastrocnemius. The morphological data supported the biochemical results pointing towards a shift from apoptosis to necrosis, which seems to corroborate the aponecrosis theory.

The effects of a high-altitude exposure were studied in six mountaineers who spent 3 weeks at an altitude range between 5,250 and 7,161 m after 1 week in an acclimatization trek (2,800-5,250 m). Blood drawn from the antecubital vein was collected at sea level 1 day before and 1 day after the expedition to analyse some haematological variables [haemoglobin (Hb), haematocrit (Htc) and red blood cell (RBC) count], erythrocyte antioxidant enzyme activity [superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase (Gr)] and membrane fatty acid profile [mono-unsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), saturated fatty acids (SFA), trans fatty acids (TRANS)]. Moreover, total antioxidant status (TAS), thiobarbituric acid reactive substances (TBARS), thiol protein groups (SH), SOD, GPx and Gr were measured in plasma. High-altitude exposure induced polycythaemia, with significant increases in RBC count (5.26%), Hb concentration (4.83%) and Htc (6.26%). Furthermore, a significant increase in plasma TBARS, SOD and Gr was observed after the expedition, whereas SH, TAS and GPx decreased. Erythrocyte glutathione-cycle-related antioxidant enzyme activity was upregulated, whereas SOD activity was maintained after the expedition. In addition, despite the unchanged (MUFA+PUFA)/SFA ratio, the membrane erythrocyte fatty acid content showed a significant increase in PUFAs and a decrease in TRANS, suggesting enhanced membrane fluidity. In conclusion, it seems that high-altitude exposure, besides quantitative variations in RBC expression, induced plasma oxidative stress and damage, and significant changes in erythrocyte components, namely in antioxidant enzyme activity and membrane fatty acid profile that might modify RBC functionality.

Background: During cerebral inflammation uracil nucleotides leak to the extracellular medium and activate glial pyrimidine receptors contributing to the development of a reactive phenotype. Chronically activated microglia acquire an anti-inflammatory phenotype that favors neuronal differentiation, but the impact of these microglia on astrogliosis is unknown. We investigated the contribution of pyrimidine receptors to microglia-astrocyte signaling in a chronic model of inflammation and its impact on astrogliosis. Methods: Co-cultures of astrocytes and microglia were chronically treated with lipopolysaccharide (LPS) and incubated with uracil nucleotides for 48 h. The effect of nucleotides was evaluated in methyl-[3H]-thymidine incorporation. Western blot and immunofluorescence was performed to detect the expression of P2Y(6) receptors and the inducible form of nitric oxide synthase (iNOS). Nitric oxide (NO) release was quantified through Griess reaction. Cell death was also investigated by the LDH assay and by the TUNEL assay or Hoechst 33258 staining. Results: UTP, UDP (0.001 to 1 mM) or PSB 0474 (0.01 to 10 mu M) inhibited cell proliferation up to 43 +/- 2% (n = 10, P < 0.05), an effect prevented by the selective P2Y(6) receptor antagonist MRS 2578 (1 mu M). UTP was rapidly metabolized into UDP, which had a longer half-life. The inhibitory effect of UDP (1 mM) was abolished by phospholipase C (PLC), protein kinase C (PKC) and nitric oxide synthase (NOS) inhibitors. Both UDP (1 mM) and PSB 0474 (10 mu M) increased NO release up to 199 +/- 20% (n = 4, P < 0.05), an effect dependent on P2Y(6) receptors-PLC-PKC pathway activation, indicating that this pathway mediates NO release. Western blot and immunocytochemistry analysis indicated that P2Y(6) receptors were expressed in the cultures being mainly localized in microglia. Moreover, the expression of iNOS was mainly observed in microglia and was upregulated by UDP (1 mM) or PSB 0474 (10 mu M). UDP-mediated NO release induced apoptosis in astrocytes, but not in microglia. Conclusions: In LPS treated co-cultures of astrocytes and microglia, UTP is rapidly converted into UDP, which activates P2Y6 receptors inducing the release of NO by microglia that causes astrocyte apoptosis, thus controlling their rate of proliferation and preventing an excessive astrogliosis.