Isolation of Relaxing Particles from Rat Skeletal Muscles in Zonal Centrifuges

Supernatants of rat skeletal muscle homogenates were fractionated by differential centrifugation and by zonal centrifugation in sucrose density gradients. Cytochrome oxidase was employed as an enzymatic marker for locating mitochondria. The subcellular fractions were also assayed for their ability to prevent the ATP-induced contraction of myofibrils. Both the mitochondrial and microsomal fractions obtained by differential fractionation were found to be rich in such relaxing activity, and the microsomal fraction was appreciably contaminated by mitochondria. In contrast to this, when fractionation was carried out by means of zonal centrifugation (4200 RPM x 205 min. to 40,000 RPM x 60 min.), relaxing activity was found to be associated only with particles having the sedimentation characteristics of microsomes (s _20,w estimated to be between 370 and 1880S). Relaxing activity was not detected in the regions of the gradient containing either the starting sample zone (soluble phase) or the mitochondrial peak. The microsomal relaxing particles showed negligible cytochrome oxidase activity.     

Increasing Exercise Intensity Reduces Heterogeneity of Glucose Uptake in Human Skeletal Muscles

Proper muscle activation is a key feature of survival in different tasks in daily life as well as sports performance, but can be impaired in elderly and in diseases. Therefore it is also clinically important to better understand the phenomenon that can be elucidated in humans non-invasively by positron emission tomography (PET) with measurements of spatial heterogeneity of glucose uptake within and among muscles during exercise. We studied six healthy young men during 35 minutes of cycling at relative intensities of 30% (low), 55% (moderate), and 75% (high) of maximal oxygen consumption on three separate days. Glucose uptake in the quadriceps femoris muscle group (QF), the main force producing muscle group in recreational cycling, and its four individual muscles, was directly measured using PET and 18F-fluoro-deoxy-glucose. Within-muscle heterogeneity was determined by calculating the coefficient of variance (CV) of glucose uptake in PET image voxels within the muscle of interest, and among-muscles heterogeneity of glucose uptake in QF was expressed as CV of the mean glucose uptake values of its separate muscles. With increasing intensity, within-muscle heterogeneity decreased in the entire QF as well as within its all four individual parts. Among-muscles glucose uptake heterogeneity also decreased with increasing intensity. However, mean glucose uptake was consistently lower and heterogeneity higher in rectus femoris muscle that is known to consist of the highest percentage of fast twitch type II fibers, compared to the other three QF muscles. In conclusion, these results show that in addition to increased contribution of distinct muscle parts, with increases in exercise intensity there is also an enhanced recruitment of muscle fibers within all of the four heads of QF, despite established differences in muscle-part specific fiber type distributions. Glucose uptake heterogeneity may serve as a useful non-invasive tool to elucidate muscle activation in aging and diseased populations.     

Re-Evaluation of Sarcolemma Injury and Muscle Swelling in Human Skeletal Muscles after Eccentric Exercise

The results regarding the effects of unaccustomed eccentric exercise on muscle tissue are often conflicting and the aetiology of delayed onset muscle soreness (DOMS) induced by eccentric exercise is still unclear. This study aimed to re-evaluate the paradigm of muscular alterations with regard to muscle sarcolemma integrity and fibre swelling in human muscles after voluntary eccentric exercise leading to DOMS. Ten young males performed eccentric exercise by downstairs running. Biopsies from the soleus muscle were obtained from 6 non-exercising controls, 4 exercised subjects within 1 hour and 6 exercised subjects at 2–3 days and 7–8 days after the exercise. Muscle fibre sarcolemma integrity, infiltration of inflammatory cells and changes in fibre size and fibre phenotype composition as well as capillary supply were examined with specific antibodies using enzyme histochemistry and immunohistochemistry. Although all exercised subjects experienced DOMS which peaked between 1.5 to 2.5 days post exercise, no significant sarcolemma injury or inflammation was detected in any post exercise group. The results do not support the prevailing hypothesis that eccentric exercise causes an initial sarcolemma injury which leads to subsequent inflammation after eccentric exercise. The fibre size was 24% larger at 7–8 days than at 2–3 days post exercise (p<0.05). In contrast, the value of capillary number per fibre area tended to decrease from 2–3 days to 7–8 days post exercise (lower in 5 of the 6 subjects at 7–8 days than at 2–3 days; p<0.05). Thus, the increased fibre size at 7–8 days post exercise was interpreted to reflect fibre swelling. Because the fibre swelling did not appear at the time that DOMS peaked (between 1.5 to 2.5 days post exercise), we concluded that fibre swelling in the soleus muscle is not directly associated with the symptom of DOMS.     

Effects of Acute Swimming Exercise on Some Elements in Rats

The objective of the present study is to explore the effects of acute swimming exercise on plasma levels of some elements in rats, immediately after the exercise, and 24 and 48 h later. The study included 40 adult male rats of Spraque Dawley species, which were equally allocated to four groups. Group 1: General Control Group; Group 2: Swimming Group, the group that was decapitated immediately after 30-min acute swimming exercise; Group 3: Swimming Group, the group that was decapitated 24 h after 30-min acute swimming exercise; Group 4: Swimming Group, the group that was decapitated 48 h after 30-min acute swimming exercise. Plasma copper (Cu), iron (Fe), magnesium (Mg), phosphorus (P), selenium (Se), zinc (Zn) levels were determined according to atomic emission method in the blood samples collected from the animals by decapitation method. Measurements conducted immediately after acute swimming exercise (group 2) showed a significant decrease in Se and Zn levels (p < 0,01) and a significant increase in P levels (p < 0,01), when compared to group 1. Measurements carried out 24 h after the exercise (group 3) demonstrated a significant increase in all parameters except for Mg, in comparison to groups 1 and 2 (p < 0,01). It was seen in the measurements made 48 h after the exercise (group 4) that all parameters were restored to control values. The results of our study show that acute swimming exercise significantly changes plasma Cu, Fe, P, Se, and Zn levels.     

Mitochondrial OXPHOS Functions in R1H Rhabdomyosarcoma and Skeletal Muscles of the Rat

The aim of the study was to determinate mitochondrial oxidative phosphorylation (OXPHOS) functions in rat rhabdomyosarcoma R1H (R1H) and rat skeletal muscles. For that purpose skinned fiber technique and multiple substrate inhibitor titration were adapted to tumor samples. In our animal tumor model (R1H) functional abnormalities of OXPHOS were found compared to skeletal muscles. In R1H the state 3 respiration of pyruvate + malate was decreased: 0.56 +/- 0.28 nmol O(2)/mg/min versus 2.32 +/- 1.19 nmol O(2)/mg/min, P < 0.001, whereas the state 3 respiration of succinate + rotenone was increased: 36 +/- 14% versus 19 +/- 11%, P < 0.001. In R1H the rotenone-insensitive respiration reached higher levels than the antimycin A-insensitive respiration, whereas in normal muscles the converse was observed. Additionally, the obvious difference between the CAT- and the antimycin A-independent respiration indicates an increased part of leak respiration in R1H. By now, the high feasibility of these techniques is appreciated for the investigation of muscles and prospectively for tumors, too.     

Differential Proteome Analysis of Hagfish Dental and Somatic Skeletal Muscles

Hagfish, the plesiomorphic sister group of all vertebrates, are deep-sea scavengers. The large musculus (m.) longitudinalis linguae (dental muscle) is a specialized element of the feeding apparatus that facilitates the efficient ingestion of food. In this article, we compare the protein expression in hagfish dental and somatic (the m. parietalis) skeletal muscles via two-dimensional gel electrophoresis and mass spectrometry in order to characterize the former muscle. Of the 500 proteins screened, 24 were identified with significant differential expression between these muscles. The proteins that were overexpressed in the dental muscle compared to the somatic muscle were troponin C (TnC), glycogen phosphorylase, β-enolase, fructose-bisphosphate aldolase A (aldolase A), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In contrast, myosin light chain 1 (MLC 1) and creatine kinase (CK) were over-expressed in the somatic muscle relative to the dental muscle. These results suggest that these two muscles have different energy sources and contractile properties and provide an initial representative map for comparative studies of muscle-protein expression in low craniates.     

Adaptive Characteristics of Skiers with Different Types of Energy Supply of Skeletal Muscles to Graded Exercise and Athletic Training

Thirty-five skiers with different types of energy supply to skeletal muscle were studied over a one-year training cycle. The adaptive characteristics of the adolescent boys were assessed by Baevskii's index of stress in the body's regulatory systems (stress index) caused by exercise at high workloads (3 and 6 W/kg); by the physiological cost of the exercise; by the time to exhaustion at a moderate workload (1.5 W/kg) in a bicycle ergometer test; and by athletic results. The stress index, the physiological cost of exercise and their changes over a one-year training cycle were clearly correlated with the type of energy production. It was suggested that training brings into play genetically determined adaptive programs and that an increase in the functional capabilities during ontogenesis is under control of the genome and mainly determined by individual and typological features of the body.     

Heterogeneous Effects of Calorie Restriction on In Vivo Glucose Uptake and Insulin Signaling of Individual Rat Skeletal Muscles

Calorie restriction (CR) (consuming ∼60% of ad libitum, AL, intake) improves whole body insulin sensitivity and enhances insulin-stimulated glucose uptake by isolated skeletal muscles. However, little is known about CR-effects on in vivo glucose uptake and insulin signaling in muscle. Accordingly, 9-month-old male AL and CR (initiated when 3-months-old) Fischer 344xBrown Norway rats were studied using a euglycemic-hyperinsulinemic clamp with plasma insulin elevated to a similar level (∼140 µU/ml) in each diet group. Glucose uptake (assessed by infusion of [¹⁴C]-2-deoxyglucose, 2-DG), phosphorylation of key insulin signaling proteins (insulin receptor, Akt and Akt substrate of 160kDa, AS160), abundance of GLUT4 and hexokinase proteins, and muscle fiber type composition (myosin heavy chain, MHC, isoform percentages) were determined in four predominantly fast-twitch (epitrochlearis, gastrocnemius, tibialis anterior, plantaris) and two predominantly slow-twitch (soleus, adductor longus) muscles. CR did not result in greater GLUT4 or hexokinase abundance in any of the muscles, and there were no significant diet-related effects on percentages of MHC isoforms. Glucose infusion was greater for CR versus AL rats (P<0.05) concomitant with significantly (P<0.05) elevated 2-DG uptake in 3 of the 4 fast-twitch muscles (epitrochlearis, gastrocnemius, tibialis anterior), without a significant diet-effect on 2-DG uptake by the plantaris or either slow-twitch muscle. Each of the muscles with a CR-related increase in 2-DG uptake was also characterized by significant (P<0.05) increases in phosphorylation of both Akt and AS160. Among the 3 muscles without a CR-related increase in glucose uptake, only the soleus had significant (P<0.05) CR-related increases in Akt and AS160 phosphorylation. The current data revealed that CR leads to greater whole body glucose disposal in part attributable to elevated in vivo insulin-stimulated glucose uptake by fast-twitch muscles. The results also demonstrated that CR does not uniformly enhance either insulin signaling or insulin-stimulated glucose uptake in all muscles in vivo.     

早期游泳运动对慢性温和应激抑郁大鼠学习和记忆能力的影响

目的:探讨早期游泳运动对慢性温和应激(chronic mild stress,CMS)大鼠学习和记忆能力的影响。方法:50只SD大鼠随机分成5组:对照组、模型组、低强度运动组、中等强度运动组、高强度运动组。运动组大鼠按照游泳运动方案运动4 w,糖水消耗实验和强迫游泳实验评价大鼠抑郁程度;Morris水迷宫实验评价大鼠学习记忆能力。结果:模型组大鼠糖水消耗量和游泳不动时间较对照组明显降低(P0.01),低强度运动组和中等强度运动组大鼠较模型组糖水消耗量和游泳不动时间明显增加(P0.05或P0.01);模型组大鼠较对照组逃避潜伏期明显延长,目标象限搜索时间占总游泳时间的百分比和目标象限穿过次数减少,体质量、双侧海马质量和海马相对质量下降(P0.05或P0.01);低强度运动组和中等强度运动组大鼠较模型组比较,潜伏期缩短明显,目标象限搜索时间占总游泳时间的百分比和目标象限穿过次数增加,体质量、双侧海马质量和海马相对质量增加(P0.05或P0.01)。结论:早期游泳运动能减轻慢性温和应激抑郁大鼠的学习记忆的损害,其中低强度和中等强度运动训练作用效果最佳。     

Proteomic Analysis of Membrane-Associated Proteins from Rat Liver Autophagosomes

Proteins associated with membranes from purified rat liver autophagosomes were separated by two-dimensional (2D) gel electrophoresis (zoom gels, pI 4-7 and 6-9), silver-stained and identified by MALDI-TOF mass spectrometry. Among >1,500 detectable protein spots, 58 (derived from 39 different known proteins) were at least twofold (and significantly) enriched in autophagosomal membranes relative to cytoplasmic membranes. All of these membrane-associated proteins were also present in the cytosol, many of them being truncated enzyme variants that would be expected to serve a binding rather than an enzymatic function.     

Chloride Currents in Skeletal Muscles of Bufo arenarum

Cl⁻ currents (I _Cl) were measured in short fibers (1–2 mm) from the lumbricalis muscle of toads (Bufo arenarum) with two microelectrodes (15°C). Initially the fibers were equilibrated in a high K⁺-containing solution: (mm) K₂SO₄ 68; Na₂SO₄ 20; KCl 60; CaSO₄ 8; MgSO₄ 1; HEPES 2.5. Constant pulses were applied when all the external K⁺ was replaced by Cs⁺: Cs₂SO₄ 68; Na₂SO₄ 20; CsCl 60; CaSO₄ 8; HEPES 2.5 (pH 7.5). Under these conditions about 80–90% of the current is carried by Cl⁻. The current-voltage relation is almost linear implying constant conductance and hence voltage-independent permeability. The voltage dependence of the net Cl⁻ current could be fitted by constant field equation with a P _Cl of 3.3 × 10⁻⁶ cm/sec. In a separate group of experiments a two-pulse technique was used to estimate the availability and the inactivation of the initial I _Cl during a test pulse. After returning the potential to the holding potential for various times, test pulses of the same amplitude and duration of the prepulses were applied. The initial current during the test pulse was 70% of the initial current during the prepulse and the recovery was complete in less than 300 msec with a linear relationship between the current during the test pulse and the amplitude of the preceding prepulse. When the test pulses were preceded by a positive prepulse, the initial current for any given test pulse was larger than with a negative prepulse. If we assumed that the initial current during the test pulse is a measure of the number of channels open at the end of the prepulse, these results suggest that hyperpolarizing pulses inactivate and depolarizing prepulses activate the I _Cl. Received: 31 March 1995/Revised: 27 October 1995     

Activation of Nrf2 by SFN Improves Hypoxic Exercise Endurance and Lactate Metabolism in Red and White Skeletal Muscles of Mice

Exercise endurance is closely related to the production and elimination of lactate in skeletal muscles. It has been confirmed in literature that nuclear factor erythroid-2-related factor 2 (Nrf2) plays an important role in exercise-induced skeletal muscle antioxidant, mitochondrial biosynthesis and energy metabolism. However, it is still unclear whether Nrf2 activation has any effect on lactate metabolism and exercise endurance under hypoxic environment. In this study, sulforaphane (SFN), the Nrf2 activator, was pretreated to mice and then the mice were subjected to an exhaustive exercise under hypoxia (11.2% O₂). The results showed that the exhaustive exercise significantly increased the blood lactate level in PBS-exercise group (P<0.05). However, this indicator in the SFN-exercise group was only 76% of that in the PBS-exercise group (P<0.05). Also, the running distance increased from 577.0 ± 52.9 m to 636.3 ± 101.4 m and the duration increased by 1.1 times as well (P<0.05). Western blotting was used to determine the relative expression of proteins in skeletal muscles. Protein expression levels of Nrf2 and p-Nrf2 in the SFN-control and SFN-exercise group were significantly increased in soleus (P<0.05). In extensor digitorum longus, SFN pretreatment induced the expression of p-Nrf2 increased by 38% and 52%, respectively (P<0.05). In addition, SFN pretreatment promoted MCT1 protein expression in SFN-Control/ SFN-Exercise group and decreased MCT4 protein expression level in SFN-Exercise group (P<0.05). Meanwhile, the expression of LDH-B proteins in soleus was increased by 62% (P<0.05). In extensor digitorum longus, expression of LDH-B in the SFN-control and SFN-exercise group was increased by 1.35 times and 1.31 times, respectively (P<0.05). These results suggested that activation of Nrf2 by SFN could improve the lactate transport and metabolism capacity of skeletal muscle, which probably was one of the potential reasons for the increased exercise endurance of mice in the hypoxic environment. This study provided some theoretical reference for the practical application of SFN in athletes, mountaineers and highland workers.     

大鼠海马细胞质膜的分离及其蛋白质组学分析

运用差速离心和连续蔗糖密度梯度离心的方法分离纯化成年大鼠海马组织细胞质膜并用Western blotting对质膜样品进行检测.结果表明,在蔗糖密度梯度离心介质中,膜片主要集中于蔗糖浓度为32%~42%的区段(密度约1.13~1.18),其次是20%~25% (密度约1.08~1.10)的区段.39%(密度约1.17)层面上质膜浓度和纯度最高.一维SDS-PAGE分离和CapLC-MS/MS分析后,通过数据库搜寻从大鼠海马组织细胞质膜样品中共鉴定出135种蛋白质, 其中质膜蛋白和与膜相关的蛋白质共70种(占51.9%), 主要包括Na⁺/K⁺-ATPase、Glutamate/aspartate transporter、Lipophilin、GLAST 1a等. 为研究海马组织细胞的功能和大鼠海马组织细胞质膜蛋白质数据库的建立积累了有价值的资料.