Effect of oral glutamine supplementation on resting blood and saliva immune parameters in endurance trained athletes

There is considerable evidence suggesting that frequent, intense, long duration exercise is associated with adverse effects on the immune system as shown by a decrease in immune cell numbers and function. This may ultimately be responsible for the increased rate of infection, in particular, upper respiratory tract infection, seen in athletes. It has been suggested that one possible mechanism for this immunosuppression is a decrease in plasma glutamine. Glutamine is a non-essential amino acid that is utilized at a high rate by immune cells. A decrease in plasma glutamine may then impair immune function and possibly lead to increased rates o f infection. Recent research has shown that plasma glutamine levels are decreased during times of metabolic and catabolic stress, such as cancer, burns, surgery, endurance exercise and overtiaiiung. This decrease in plasma glutamine coupled with the immunocompronused state of these individuals may be evidence of the important role of glutamine in immune function Recent research has shown that glutamine supplementation results in increased immune cell counts and immune function, and decreased rates of infection in various populations. Therefore, the purpose of this study was to determine the effect of oral glutamine supplementation on resting blood and saliva immune parameters in endurance trained athletes. Fifteen endurance athletes (male= 9, female = 6, age = 27.9 ± 2.2 yrs, height = 177.0 ±2.1 cm, weight=68.5 ± 2.8 kg, running = 64.1 ± 3.2 km/wk) participated in a randomized double-blind glutamine/placebo cross-over study. Thirty grams of glutamine or placebo was provided for a duration of 2 weeks, separated by a 4 week washout Resting blood and saliva was collected before and after each supplementation period and was analyzed for, total leukocytes, neutrophils, monocytes, lymphocytes, T cells, B cells, natural killer, CD4, CD8 cells, saliva IgA and plasma glutamine using a 2-way repeated measures factorial ANOVA. The effect of gender was analyzed by a 3-way mixed plot factorial ANOVA. As well, subjects completed a nutritional analysis, a life stress questionnaire and a training log during the supplementation periods in order to monitor any changes in these control variables. Statistical analysis revealed that leukocyte and neutrophil count as well as percent neutrophils for female subjects significantly increased by 13.7 %, 33.5 %, and 16.1 %, respectively following glutamine supplementation, while those receiving placebo decreased by 132 %, 23.2 %, and 10.5 %, respectively (p0.05) in any immune parameters of male athletes. This data suggests that under these conditions oral glutamine supplementation may be an effective method of increasing leukocyte and neutrophil counts in endurance trained females

Rapid Determination of Myosin Heavy Chain Expression in Rat, Mouse, and Human Skeletal Muscle Using Multicolor Immunofluorescence Analysis

Skeletal muscle is a heterogeneous tissue comprised of fibers with different morphological, functional, and metabolic properties. Different muscles contain varying proportions of fiber types; therefore, accurate identification is important. A number of histochemical methods are used to determine muscle fiber type; however, these techniques have several disadvantages. Immunofluorescence analysis is a sensitive method that allows for simultaneous evaluation of multiple MHC isoforms on a large number of fibers on a single cross-section, and offers a more precise means of identifying fiber types. In this investigation we characterized pure and hybrid fiber type distribution in 10 rat and 10 mouse skeletal muscles, as well as human vastus lateralis (VL) using multicolor immunofluorescence analysis. In addition, we determined fiber type-specific cross-sectional area (CSA), succinate dehydrogenase (SDH) activity, and α-glycerophosphate dehydrogenase (GPD) activity. Using this procedure we were able to easily identify pure and hybrid fiber populations in rat, mouse, and human muscle. Hybrid fibers were identified in all species and made up a significant portion of the total population in some rat and mouse muscles. For example, rat mixed gastrocnemius (MG) contained 12.2% hybrid fibers whereas mouse white tibialis anterior (WTA) contained 12.1% hybrid fibers. Collectively, we outline a simple and time-efficient method for determining MHC expression in skeletal muscle of multiple species. In addition, we provide a useful resource of the pure and hybrid fiber type distribution, fiber CSA, and relative fiber type-specific SDH and GPD activity in a number of rat and mouse muscles

Fibre-Specific Responses to Endurance and Low Volume High Intensity Interval Training: Striking Similarities in Acute and Chronic Adaptation

The current study involved the completion of two distinct experiments. Experiment 1 compared fibre specific and whole muscle responses to acute bouts of either low-volume high-intensity interval training (LV-HIT) or moderate-intensity continuous endurance exercise (END) in a randomized crossover design. Experiment 2 examined the impact of a six-week training intervention (END or LV-HIT; 4 days/week), on whole body and skeletal muscle fibre specific markers of aerobic and anaerobic capacity. Six recreationally active men (Age: 20.7±3.8 yrs; VO2peak: 51.9±5.1 mL/kg/min) reported to the lab on two separate occasions for experiment 1. Following a muscle biopsy taken in a fasted state, participants completed an acute bout of each exercise protocol (LV-HIT: 8, 20-second intervals at ∼170% of VO2peak separated by 10 seconds of rest; END: 30 minutes at ∼65% of VO2peak), immediately followed by a muscle biopsy. Glycogen content of type I and IIA fibres was significantly (p<0.05) reduced, while p-ACC was significantly increased (p<0.05) following both protocols. Nineteen recreationally active males (n = 16) and females (n = 3) were VO2peak-matched and assigned to either the LV-HIT (n = 10; 21±2 yrs) or END (n = 9; 20.7±3.8 yrs) group for experiment 2. After 6 weeks, both training protocols induced comparable increases in aerobic capacity (END: Pre: 48.3±6.0, Mid: 51.8±6.0, Post: 55.0±6.3 mL/kg/min LV-HIT: Pre: 47.9±8.1, Mid: 50.4±7.4, Post: 54.7±7.6 mL/kg/min), fibre-type specific oxidative and glycolytic capacity, glycogen and IMTG stores, and whole-muscle capillary density. Interestingly, only LV-HIT induced greater improvements in anaerobic performance and estimated whole-muscle glycolytic capacity. These results suggest that 30 minutes of END exercise at ∼65% VO2peak or 4 minutes of LV-HIT at ∼170% VO2peak induce comparable changes in the intra-myocellular environment (glycogen content and signaling activation); correspondingly, training-induced adaptations resulting for these protocols, and other HIT and END protocols are strikingly similar

Caspase activity and apoptotic signaling in proliferating C2C12 cells following cisplatin or A23187 exposure

Investigating cell death signaling using cell culture is commonly performed to examine the effects of novel pharmaceuticals or to further characterize discrete cellular signaling pathways. Here, we provide data regarding the cell death response to either cisplatin or A23187 in sub-confluent C2C12 cells, by utilizing several concentrations and incubation times for each chemical. These data include an assessment of the activation of the proteolytic enzymes caspase-3, caspase-8, caspase-9, calpain, and cathepsin B/L. Additionally, the expression of the apoptosis-regulating proteins Bax, Bcl2, and p53 are presented

Autophagic flux data in differentiated C2C12 myotubes following exposure to acetylcholine and caffeine

The C2C12 line of mouse myoblasts is a useful cell culture model in which to conduct in vitro analyses related to skeletal muscle. Here we present data regarding the autophagic response induced by two chemicals known to influence calcium release and contraction in skeletal muscles and C2C12 cells: acetylcholine and caffeine. More specifically, by concurrently administering acetylcholine or caffeine along with chloroquine to differentiated myotubes for various amounts of time and assessing the protein expression of LC3 and p62, we report data on the relative level of autophagic flux induced by these two calcium- and contraction-regulating chemicals. Keywords: Skeletal muscle, Autophagy, Acetylcholine, Calcium, Caffeine, Myotubes, C2C1

Effect of mitochondrial fission inhibition on C2C12 differentiation

The differentiation of skeletal muscle is commonly examined in cell culture using the C2C12 line of mouse skeletal myoblasts. This process shares many similarities with that which occurs during embryonic development, such as the transient activation of caspases. Here, we examined the effect of inhibiting mitochondrial fission, using mdivi-1, on the ability of C2C12 cells to terminally differentiate. This was performed using immunofluorescent identification of cell morphology and myosin expression, as well as immunoblotting for markers of muscle differentiation. Furthermore, the effect of mdivi-1 administration on activation of caspase-2 and -3 was assessed using spectrofluorometric measurement of specific enzyme activity. Keywords: Skeletal muscle, Mitochondrial fission, Apoptosis, Differentiation, Caspases, C2C1

Data on skeletal muscle apoptosis, autophagy, and morphology in mice treated with doxorubicin

Skeletal muscle apoptosis and autophagy are catabolic processes that contribute to muscle atrophy during aging, disease, and following muscle injury. In this article, we present data on skeletal muscle apoptosis, autophagy, and morphology in C57BL/6 mice following doxorubicin administration. More specifically, time-course data on caspase-3, caspase-8, caspase-9, calpain, and cathepsin activity are presented, along with data on ATG7, p62, LC3-I, and LC3-II protein expression. Data on skeletal muscle reactive oxygen species (ROS) production, muscle morphology, as well as body and muscle weights are also presented. Keywords: Skeletal muscle, Apoptosis, Autophagy, Doxorubici

Overview of immunofluorescence MHC staining protocol.

*<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035273#pone-0035273-t001" target="_blank">Table 1</a> for primary and secondary antibody cocktail configurations.</p

Representative images of mouse red gastrocnemius (RG) muscle showing MHC expression as well as SDH and GPD activity staining.

<p>Panel A, mouse muscle serial cross-section incubated with a primary antibody cocktail against MHCI (BA-F8), MHCIIa (SC-71), and MHCIIb (BF-F3), followed by incubation with appropriate fluorescent-conjugated secondary antibodies. Shown are type I (blue), type IIA (green), type IIB (red), type IIX (unstained), and type IIXB (intermediate red) fibers. Panel B, mouse muscle serial cross-section incubated with a primary antibody cocktail against MHCIIa (SC-71) and MHCIIx (6H1), followed by incubation with appropriate fluorescent-conjugated secondary antibodies. This confirms the presence of type IIA (green) fibers, as well as confirms that the unstained fibers and intermediate red stained fibers in Panel A are type IIX (purple) and type IIXB (purple and red) fibers, respectively. Panel C, mouse muscle serial cross-section showing SDH activity staining. Panel D, mouse muscle serial cross-section showing GPD activity staining. Bar represents 50 µm.</p