A Method to Induce Brown/Beige Adipocyte Differentiation from Murine Preadipocytes.

Adipocytes exhibit different morphological and functional characteristics, depending on their anatomical location, developmental origin, and stimulus. While white adipocytes tend to accumulate energy as triglycerides, brown and beige adipocytes tend to direct carbon sources to fuel thermogenesis. White and beige adipocytes originate from common progenitor cells, which are distinct from brown adipocyte precursors. Having a method to study white vs. beige vs. brown adipocyte differentiation may help to unveil the mechanisms driving distinct adipogenic programs. Preadipocytes can be cultured and differentiated in vitro using a combination of compounds to stimulate adipogenesis. Here, we describe and compare protocols designed to stimulate adipocyte differentiation and induce brown/beige-like or white-like characteristics in differentiating adipocytes. The protocols consist in exposing murine preadipocytes to pharmacological stimuli aimed at triggering adipogenesis and inducing (or not) a thermogenic gene expression program. After 8 days of differentiation with a pro-browning cocktail, immortalized preadipocytes isolated from interscapular brown fat (9B cells) or inguinal white fat (9W cells) from the same mouse expressed higher levels of brown/beige adipocyte markers (e.g., Ucp1) and pan-adipocyte differentiation markers (e.g., Pparg, Cebpa and aP2) when compared to the same cells differentiated with a cocktail that lacked brown/beige adipogenic inducers (i.e., rosiglitazone, T3, and indomethacin). Consistent with a higher thermogenic potential of brown vs. beige adipocytes, differentiated 9B cells expressed higher Ucp1 levels than differentiated 9W cells. This simple protocol may help researchers to understand mechanisms of adipogenesis and how adipocytes become thermogenic.

Oxidative stress and antioxidant status response of handball athletes: implications for sport training monitoring.

The chronic exposure to regular exercise training seems to improve antioxidant defense systems. However, the intense physical training imposed on elite athletes may lead to overtraining associated with oxidative stress. The purpose of the present study was to investigate the effect of different training loads and competition on oxidative stress, biochemical parameters and antioxidant enzymatic defense in handball athletes during 6-months of monitoring. Ten male elite handball athletes were recruited to the study. Blood samples were collected four times every six weeks throughout the season. During most intense periods of training and competitions there were significant changes in plasma indices of oxidative stress (increased TBARS and decreased thiols). Conversely, chronic adaptations to exercise training demonstrated a significant protective effect against oxidative stress in erythrocyte (decrease in TBARs and carbonyl group levels). Erythrocyte antioxidant enzyme activities were significantly increased, suggesting a training-induced antioxidant adaptation. Biomarkers of skeletal muscle damage were significantly increased during high-intensity training period (creatine kinase, lactate dehydrogenase and aspartate aminotransferase). No significant changes were observed in plasma IL-6, TNF-α and uric acid, whereas a significant reduction was found in the IL-1ß concentration and gamma-glutamyl transferase activity. Oxidative stress and antioxidant biomarkers can change throughout the season in competitive athletes, reflecting the physical stress and muscle damage that occurs as the result of competitive handball training. In addition, these biochemical measurements can be applied in the physiological follow-up of athletes.

Glycolaldehyde impairs neutrophil biochemical parameters by an oxidative and calcium-dependent mechanism--protective role of antioxidants astaxanthin and vitamin C.

AIM: The present study examined the effects of glycolaldehyde (GC) on biochemical parameters of human neutrophils and whether the antioxidant astaxanthin associated with vitamin C can modulate these parameters. METHODS: Neutrophils from healthy subjects were treated with GC (1mM) followed or not by the antioxidants astaxanthin (2 µM) and vitamin C (100 µM). We examined the phagocytic capacity, hypochlorous acid, myeloperoxidase (MPO) and glucose-6-phosphate dehydrogenase (G6PDH) activities, cytokines and [Ca(2+)](i). Also, superoxide anion, hydrogen peroxide, nitric oxide production, antioxidant enzyme activities and glutathione-recycling system were evaluated. RESULTS: GC promoted a marked reduction on the phagocytic capacity, maximal G6PDH and MPO activities, hypochlorous acid production and release of IL-1ß, IL-6 and TNF-α cytokines. Some impairment in the neutrophils biochemical parameters appears to be mediated by oxidative stress through ROS/RNS production and calcium reduction. Oxidative stress was evidenced by reduction in the activities of the main antioxidant enzymes, GSH/GSSG ratio and in the increment of O(2)(-) and H(2)O(2) and NO. CONCLUSIONS: Treatment of cells with the combination of the antioxidants astaxanthin and vitamin C was able to restore some neutrophils function mainly by decreasing ROS/RNS production and improving the redox state. Overall, our findings demonstrate that GC modulates several neutrophils biochemical parameters in vitro.

Combined astaxanthin and fish oil supplementation improves glutathione-based redox balance in rat plasma and neutrophils.

The present study aimed to investigate the effects of daily (45 days) intake of fish oil (FO; 10mg EPA/kg body weight (BW) and 7 mg DHA/kg BW) and/or natural ASTA (1mg ASTA/kg BW) on oxidative stress and functional indexes of neutrophils isolated from Wistar rats by monitoring superoxide (O(2)(-)), hydrogen peroxide (H(2)O(2)), and nitric oxide (NO()) production compared to the progression of auto-induced lipid peroxidation and Ca(2+) release in activated neutrophils. Furthermore, phagocytic capacity, antioxidant enzyme activities, glutathione-recycling system, and biomarkers of lipid and protein oxidation in neutrophils were compared to the redox status. Our results show evidence of the beneficial effects of FO+ASTA supplementation for immune competence based on the redox balance in plasma (significant increase in GSH-dependent reducing power), non-activated neutrophils (increased activity of the glutathione-recycling enzymes GPx and GR) and PMA-activated neutrophils (lower O(2)(-), H(2)O(2), and NO() generation, reduced membrane oxidation, but higher phagocytic activity). Combined application of ASTA and FO promoted hypolipidemic/hypocholesterolemic effects in plasma and resulted in increased phagocytic activity of activated neutrophils when compared with ASTA or FO applied alone. In PMA-activated neutrophils, ASTA was superior to FO in exerting antioxidant effects. The bulk of data reinforces the hypothesis that habitual consumption of marine fish (e.g. salmon, which is a natural source of both astaxanthin and fish oil) is beneficial to human health, in particular by improving immune response and lowering the risk of vascular and infectious diseases.

Combined fish oil and astaxanthin supplementation modulates rat lymphocyte function.

PURPOSE: Higher intakes of n-3 polyunsaturated fatty acids that are abundant in marine fishes have been long described as a "good nutritional intervention" with increasing clinical benefits to cardiovascular health, inflammation, mental, and neurodegenerative diseases. The present study was designed to investigate the effect of daily fish oil (FO-10 mg EPA/kg body weight (BW) and 7 mg DHA/kg BW) intake by oral gavage associated with the antioxidant astaxanthin (ASTA-1 mg/kg BW) on the redox metabolism and the functional properties of lymphocytes from rat lymph nodes. METHODS: This study was conducted by measurements of lymphocyte proliferation capacity, ROS production [superoxide (O2(•-)) and hydrogen peroxide (H2O2)], nitric oxide (NO(•)) generation, intracellular calcium release, oxidative damage to lipids and proteins, activities of major antioxidant enzymes, GSH/GSSG content, and cytokines release. RESULTS: After 45 days of FO + ASTA supplementation, the proliferation capacity of activated T- and B-lymphocytes was significantly diminished followed by lower levels of O2(•-), H2O2 and NO(•) production, and increased activities of total/SOD, GR and GPx, and calcium release in cytosol. ASTA was able to prevent oxidative modification in cell structures through the suppression of the oxidative stress condition imposed by FO. L: -selectin was increased by FO, and IL-1ß was decreased only by ASTA supplementation. CONCLUSION: We can propose that association of ASTA with FO could be a good strategy to prevent oxidative stress induced by polyunsaturated fatty acids and also to potentiate immuno-modulatory effects of FO.

Impact of the carotenoid astaxanthin on phagocytic capacity and ROS/RNS production of human neutrophils treated with free fatty acids and high glucose.

BACKGROUND: The purpose of the present study was to evaluate the effect of carotenoid astaxanthin (ASTA) on human neutrophils treated with high glucose and free fatty acids (FFA) on the phagocytic capacity and reactive oxygen/nitrogen species production. METHODS: The following parameters were evaluated: phagocytic capacity of neutrophils by using zymosan particles, intracellular and extracellular superoxide anion (lucigenin and DHE probes), hydrogen peroxide (H(2)O(2) - phenol red), nitric oxide (Griess reagent) production, and maximal activity of G6PDH. RESULTS: There was a decreased phagocytic capacity of human neutrophils treated with high glucose (30 mM) or FFA (0.1mM) and a partial restoring of the phagocytic capacity after ASTA-treatment was observed. ROS and RNS production was increased in neutrophils due to both high glucose and FFA. This increase in ROS/RNS production was also partially prevented by ASTA treatment. Both glucose and FFA increased the G6PDH activity. We show that ASTA provides a modest improvement of cellular functions after cells have been treated with high glucose and FFA. CONCLUSIONS: In summary, this study showed that both high glucose and a mixture of FFA are potent inducers of ROS/RNS production on neutrophils as observed by higher levels of superoxide anion, hydrogen peroxide and NO production. Also, these metabolites decrease the phagocytic capacity of neutrophils and increase the G6PDH activity. Overall, ASTA-treatment was able to reduce partially ROS/RNS production by reducing the availability of NADPH and recover phagocytic capacity of neutrophils.

Cytokines and oxidative stress status following a handball game in elite male players.

BACKGROUND: Handball is considered an intermittent sport that places an important stress on a player's aerobic and anaerobic metabolism. However, the oxidative stress responses following a handball game remain unknown. We investigated the responses of plasma and erythrocyte antioxidant system and oxidative stress biomarkers following a single handball game. METHODS: Fourteen male elite Brazilian handball athletes were recruited in the present study. Blood samples were taken before, immediately, and 24 hours after the game. RESULTS: After the game and during 24 hours of recovery, the concentration of all oxidative stress indices changed significantly in a way indicating increased oxidative stress in the blood (thiol groups and reduced glutathione decreased, whereas TBARS and plasma antioxidant capacity was increased) as well as in erythrocyte (increased levels of TBARS and protein carbonyls). Erythrocyte antioxidant enzyme activities were also significantly changed by handball. Muscle damage indices (creatine kinase and lactate dehydrogenase) increased significantly after exercise. In addition, IL-6 increased after the game, whereas TNF-α decreased during recovery. CONCLUSION: This study demonstrates that a single handball game in elite athletes induces a marked state of oxidative stress evidenced by the oxidative modification in plasma and erythrocyte macromolecules, as well as by changes in the enzymatic and nonenzymatic antioxidant system.