Different cells make different proteins: a laboratory exercise illustrating tissue-specific protein expression in animals

All the cells of higher organisms have the same DNA but not the same proteins. Each type of specialised cell that forms a tissue has its own pattern of gene expression and, consequently, it contains a particular set of proteins that determine its function. Here, we describe a laboratory exercise addressed to undergraduate students that aims to reinforce the following concept: what makes cells different is they make different proteins. The students performed a denaturing polyacrylamide gel electrophoresis (SDS–PAGE) to separate the proteins extracted from different animal tissues, and then obtained and compared the corresponding protein profiles. In addition, the students performed a Western blot analysis to detect the presence or absence of a tissue-specific protein in the different tissue extracts. This laboratory exercise allowed students to understand the basis of the cell differentiation better and also provided them the opportunity to learn a variety of analytical laboratory techniques.     

Rapid induction of REDD1 expression by endurance exercise in rat skeletal muscle

An acute bout of exercise induces repression of protein synthesis in skeletal muscle due in part to reduced signaling through the mammalian target of rapamycin complex 1 (mTORC1). Previous studies have shown that upregulated expression of regulated in DNA damage and development (REDD) 1 and 2 is an important mechanism in the regulation of mTORC1 activity in response to a variety of stresses. This study investigated whether induction of REDD1/2 expression occurs in rat skeletal muscle in response to a burst of endurance exercise. In addition, we determined if ingestion of glucose or branched chain amino acids (BCAA) before exercise changes the expression of REDD1/2 in muscle. Rats ran on a motor-driven treadmill at a speed of 28 m min⁻¹ for 90 min, and then the gastrocnemius muscle was removed and analyzed for phosphorylation of the eukaryotic initiation factor (eIF) 4E binding protein 1 (4E-BP1) and expression of REDD1/2. Exercise repressed the mTORC1-signaling pathway regardless of the ingestion of nutrients before the exercise, as shown by dephosphorylation of 4E-BP1. In addition, exercise induced the expression of REDD1 mRNA (∼8-fold) and protein (∼3-fold). Exercise-induced expression of REDD1 was not affected by the ingestion of glucose or BCAA. Expression of REDD2 mRNA was not altered by either exercise or nutrients. These findings indicated that enhanced expression of REDD1 may be an important mechanism that could partially explain the downregulation of mTORC1 signaling, and subsequent inhibition of protein synthesis in skeletal muscle during exercise.     

Sex differences in release of cardiac troponin T after endurance exercise

Context: Post-exercise cardiac troponin release has been extensively described in athletic groups but little attention has been given to any role of sex in mediating this phenomenon.

Objective: We compared the release of cardiac troponin T (cTnT) after endurance running in training-experience, biological-age and maturity-matched young male and female runners.

Materials and methods: Nineteen male (training history: 2.3?±?1.0?years; mean age: 16.1?±?1.2?years; Tanner stage: 3.7?±?0.6) and 19 female (training history: 2.2?±?1.0?years; mean age: 15.9?±?1.4?years; Tanner stage: 4.0?±?0.4) runners performed a 21?km run with “all-out” effort. Serum cTnT levels were assessed at pre-exercise (Pre-ex) and at 4?h post-exercise (Post-ex).

Results: At Pre-ex, cTnT concentrations were below the 99th percentile value (10?ng.l^?1) in 32/38 runners. Post-ex cTnT increased in all subjects but the response was substantially higher (p?<?0.05) in males [median (range): 210 (20–1360) ng.l^?1] than females [median (range): 80 (10–550) ng.l^?1]. At Post-ex, 95% (95% confidence interval: 75–99%) of males and 63% (95% confidence interval: 41–81%) of females (p?<?0.05) had cTnT concentrations above the cut-off for acute myocardial infarction.

Conclusions: The present data suggest that post-exercise cTnT elevation occurs in all runners but is augmented in young male compared to female athletes.     

大负荷运动诱导大鼠骨骼肌损伤对其自噬超微结构及Beclin1和LC3-II/I的影响*

目的: 观察大负荷离心运动对大鼠骨骼肌自噬超微结构及自噬相关蛋白Beclin1和LC3II/I的影响。方法: 48只SD雄性大鼠适应性训练后随机分成对照组(C,n=8)和大负荷离心运动组(E,n=40)。E组于跑台进行90 min下坡跑,运动后0 h、12 h、24 h、48 h和72 h取比目鱼肌,透射电镜观察其自噬体超微结构变化;Western blot检测Beclin1和LC3II/I蛋白表达;免疫荧光观测LC3的定位及含量变化。结果: E组比目鱼肌自噬体数量在运动后0 h、12 h和24 h均有增加,并伴LC3自噬荧光明显增强(P＜0.01),同时运动后48 h自噬荧光仍有显著性升高(P＜0.05);Beclin1和LC3II/I在大负荷离心干预后表达升高(P＜0.05),运动后12 h～24 h达到峰值(P＜0.01),直至运动后72 h完全恢复。结论: 大负荷离心运动可诱导骨骼肌自噬超微结构变化,自噬蛋白表达增强,以上可能是运动损伤的骨骼肌功能下降的原因之一。     

Effects of one bout of endurance exercise on the expression of myogenin in human quadriceps muscle

The objective of this study was to investigate the cellular localisation of MyoD and myogenin in human skeletal muscle fibres as well as the possible alterations in the expression of MyoD and myogenin in response to a single bout of endurance exercise at 40% and 75% of maximum oxygen uptake (VO₂ max). Twenty-five biopsies (5 per subject) from the vastus lateralis muscle were obtained before exercise, from the exercising leg at 40% and 75% of VO₂ max and from the resting leg following these exercise bouts. The tyramide signal amplification-direct and the Vectastain ABC methods using specific monoclonal antibodies were used to determine the exact location of myogenin and MyoD, to identify muscle satellite cells and to determine myosin heavy chain (MyHC) composition. At rest, myonuclei did not express MyoD or myogenin. Following a single bout of exercise at 40% and 75% of VO₂ max, an accumulation of myogenin in myonuclei and not in satellite cells was observed in biopsies from the exercised leg but not in biopsies before exercise and from the resting leg. The number of myogenin-positive myonuclei varied among individuals indicating differences in the response to a single exercise bout. In conclusion, this immunohistochemical study showed that a rapid rearrangement of myogenin expression occurs in exercised human skeletal muscles in response to a single bout of exercise.     

Age- and training-related changes in the collagen metabolism of rat skeletal muscle

The effects of ageing and life-long endurance training on the collagen metabolism of skeletal muscle were evaluated in a longitudinal study. Wistar rats performed treadmill running 5 days a week for 2 years. The activities of collagen biosynthesis enzymes, prolyl-4-hydroxylase and galactosylhydroxylysyl glucosyltransferase, were highest in the muscles of the youngest animals, decreased up to the age of 2 months and from then on remained virtually unchanged. The enzyme activity in young animals was higher in the slow collagenous soleus muscle than in the rectus femoris muscle. The enzyme activity in the soleus muscle was higher for older trained rats than older untrained rats. The relative proportion of type I collagen increased and that of type III collagen decreased with age, suggesting a more marked contribution by type I collagen to the age-related accumulation of total muscular collagen. The results show that collagen biosynthesis decreases with maturation and that life-long endurance training maintains a higher level of biosynthesis in slow muscles.     

Modulation of paraoxonase 1 and 3 expression after moderate exercise training in the rat

Paraoxonases (PONs) are a small family of antioxidant enzymes whose antiatherogenic activity is well known. The aim of the present study was the evaluation of the effects of moderate aerobic training on their expression using a rat model. In order to discriminate between PON1 and PON3 enzymatic activity, we took advantage of some differences in their substrate preferences. PON1 and PON3 enzymatic activities and their protein levels were analyzed in plasma and in liver microsomes, and their mRNA levels in the liver. Exercise training did not affect PON1 expression or enzymatic activity but increased PON3 mRNA, protein levels, and enzymatic activity. Training also induced variations in plasma membrane composition, including an increase in polyunsaturated and a decrease in mono- and di-unsaturated fatty acids. On the other hand, acute exercise inhibited PON activities while increasing PON3 protein content in liver microsomes and reversing the relative composition in mono-, di-, and poly-unsaturated fatty acids, suggesting that physical stress, by altering membrane composition, may impair PON release from liver membranes. In conclusion, we documented, for the first time, the presence of PON3 in rat serum and, notably, found that the upregulation of PON3, rather than PON1, appears to be associated with physical training.     

Glyceraldehyde-3-phosphate dehydrogenase interacts with phosphorylated Akt resulting from increased blood glucose in rat cardiac muscle

Here we describe the interaction of phosphorylated ∼40 kDa protein with phosphorylated Akt which is a serine/threonine kinase resulting from increased blood glucose in rat cardiac muscle. Mass spectrometry analysis revealed that this protein was glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Furthermore, increase in Akt and GAPDH phosporylation and induction of their association were both observed after insulin stimulation in the H9c2 cell line derived from embryonic rat ventricle. Moreover, the activation of GAPDH was upregulated when the GAPDH phosphorylation was increased. Our data suggest that GAPDH phosphorylation and association with Akt by insulin treatment have some bearing on the enhancement of GAPDH activity.

Structured summary

MINT-7891324, MINT-7891304, MINT-7891314: GAPDH (uniprotkb:P04797) physically interacts (MI:0915) with Akt (uniprotkb:P47196) by anti bait coimmunoprecipitation (MI:0006)     

The effect of exercise on expression of myokine and angiogenesis mRNA in skeletal muscle of high fat diet induced obese rat

[Purpose]

The purpose of this study was to investigate the effect of regular treadmill exercise on the mRNA expressions of myokines and angiogenesis factors in the skeletal muscle of obese rats.

[Methods]

Thirty two male Sprague-Dawley rats (4weeks old) were divided into the CO (control) and HF (high fat diet) groups. Obesity was induced in the HF group by consumption of 45% high-fat diet for 15 weeks. These groups were further subdivided into training groups (COT and HFT); the training groups conducted moderate intensity treadmill training for 8 weeks. Soleus muscles were excised and analyzed by real-time quantitative PCR.

[Results]

mRNA expression of myokines, such as PGC-1α, IL-6, and IL-15, in the COT and HFT groups (which conducted regular exercise), were higher as compared with the CO and HF groups (p < 0.05). Also, the levels in the HF group were significantly lower when compared with CO group (p < 0.05). Expression of angiogenesis mRNA, namely mTOR, VEGF, and FLT1, were significantly lower in the HF group, as compared to the CO group (p < 0.05). In addition, COT group had a higher expression of mTORC1, mTORC2, VEGF and FLT mRNA, than the CO group (p < 0.05); the HFT group also had higher expressions of mTOR, VEGF and FLT1 mRNA than the HF group (p < 0.05).

[Conclusion]

These results indicate that mRNA expression of myokines was increased through the activity of muscle contraction, and it also promoted the mRNA expression of angiogenesis due to activation of mTOR. Thus, we conclude that not only under normal health conditions, but in obesity and excess nutritional circumstances also, regular exercise seems to act positively on the glycemic control and insulin sensitivity through the angiogenesis signaling pathway.     

4周中等强度有氧运动诱导大鼠心房肌蛋白质组差异表达的研究

目的：探讨中等强度有氧运动对大鼠心房肌蛋白质组及其基因差异表达的影响,为运动心脏重塑和慢性心血管疾病康复研究提供研究依据。方法：20只雄性SD大鼠按照体重随机配对分为对照组、实验组（n=10）,实验组大鼠每次按照速度24 m·min^-1、持续训练40 min （负荷强度相当于60%~70% VO_2max）,每周训练6 d,持续训练4周中等强度有氧运动。应用双向凝胶电泳技术（2-DE）分离心房肌蛋白质点,串联飞行时间质谱仪技术鉴定电泳结果中表达量上调≥5倍以上,下调至1/5以下的13个备选目标蛋白质点。并对其中6个目标蛋白质用逆转录-聚合酶链式反应（RT-PCR）技术检测其mRNA。结果：通过软件分析,实验组与对照组比较,其中表达量下调至20%以下的点8个,上调5倍及以上点有5个,质谱鉴定分析其中的13个蛋白质点,最终鉴定出8种蛋白质和一个分子量为54 KDa的未知蛋白,包括：丙酮酸脱氢酶E1α1、线粒体乌头酸水合酶、蛋白质二硫键异构酶A3、甲基丙二酸半醛脱氢酶、线粒体二氢硫辛酸脱氢酶、异戊酰辅酶A脱氢酶、谷胱甘肽合成酶、丝裂素活化蛋白激酶3等。RT-PCR检测结果表明,与对照组相比,4周中等强度有氧运动后,大鼠心房肌中甲基丙二酸半醛脱氢酶的mRNA表达量降低（P﹤0.05）,线粒体二氢硫辛酸脱氢酶、蛋白质二硫键异构酶A3、线粒体乌头酸水合酶、谷胱甘肽合成酶的mRNA表达量降低（P>0.05）;异戊烯辅酶A脱氢酶的mRNA表达量增高（P>0.05）,表明mRNA表达水平与质谱鉴定结果的变化不完全一致。结论：4周的中等强度有氧运动诱导大鼠心房肌蛋白质组发生显著变化,有13个明显变化的目标蛋白,多数为能量物质代谢酶,这些目标蛋白质的变化与其mRNA表达量的变化并不完全一致,表明中等强度运动可能影响这些目标蛋白质上游基因转录的调控,也可影响下游翻译﹑修饰等的调控,导致表达的差异变化。     

The effects of a single bout pilates exercise on mRNA expression of bone metabolic cytokines in osteopenia women

[Purpose]

The purpose of this study was to examine the effect of a single bout pilates exercise on mRNA expression of bone metabolic cytokines in elderly osteopenia women.

[Methods]

We selected 11 people of elderly osteopenia women and loaded a single bout pilates exercise about RPE 10-14 level. The blood samples were collected before, immediately after and 60 minute after pilates exercise, then examined calcium metabolic markers in serum and extracted peripheral blood mononuclear cell (PBMC) from whole blood and confirmed mRNA expression of bone metabolic cytokines from PBMC. To clarify the changes during exercise, we designed repeated measure ANOVA as the control group to perform blood sampling without exercise.

[Results]

As a result, serum P showed significant interaction effect between group and time (p<.001), the pilates exercise group decreased about 9% at immediately after exercise and 13% during recovery after exercise (p<.05), while the control group showed a tendency to increase. Serum CK also showed a significant interaction between group and time (p<.05), the pilates group significantly increased at immediately after exercise and during recovery after exercise (p<.05) but the control group didn’t have changes. TNF-α and IL-6 mRNA expression in PBMC was significantly increased in the pilates group (p<.01, p<.05), although INF-γ mRNA expression didn’t show statistically significant difference, it tended to increase in the pilates group (NS).

[Conclusion]

These results suggested that a single bout pilates exercise of elderly osteopenia women cause hypophosphatemia with temporary muscle damage, and it leading high turnover bone metabolic state with to activate both of bone formation and bone resorption.     

Effects of acute bouts of running and swimming exercise on PGC-1alpha protein expression in rat epitrochlearis and soleus muscle

The purpose of this study was to elucidate the mechanisms underlying low-intensity exercise-induced peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) protein expression in rat skeletal muscles. Rats (5-6 wk old) swam without a load and ran on the treadmill at a speed of 13 m/min, respectively, in two 3-h sessions separated by 45 min of rest. PGC-1alpha content in epitrochlearis muscle (EPI) was increased by 75 and 95%, immediately and 6 h after swimming, respectively, with no increase in PGC-1alpha content in the soleus (SOL). After running, PGC-1alpha content in EPI was unchanged, whereas a 107% increase in PGC-1alpha content was observed in SOL 6 h after running. Furthermore, in EPI and SOL as well as other muscles (triceps, plantaris, red and white gastrocnemius), PGC-1alpha expression was enhanced concomitant with reduced glycogen postexercise, suggesting that expression of PGC-1alpha occurs in skeletal muscle recruited during exercise. PGC-1alpha content in EPI was increased after 18-h in vitro incubation with 0.5 mM 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) and 4 mM caffeine. However, AICAR incubation did not affect PGC-1alpha content in the SOL, whereas caffeine incubation increased it. These results suggest that exercise-induced PGC-1alpha expression in skeletal muscle may be mediated by at least two exercise-induced signaling factors: AMPK activation and Ca2+ elevation. The number of factors involved (both AMPK and Ca2+, or Ca2+ only) in exercise-induced PGC-1alpha expression may differ among muscles.     

A high-whey-protein diet does not enhance mechanical and structural remodeling of cardiac muscle in response to aerobic exercise in rats

[Purpose]Aerobic exercise training results in distinct structural and mechanical myocardial adaptations. In skeletal muscle, whey protein supplementation is effective in enhancing muscle adaptation following resistance exercise. However, it is unclear whether cardiac adaptation to aerobic exercise can be enhanced by systematic protein supplementation.[Methods]Twelve-week-old rats were assigned to 12 weeks of either sedentary or aerobic exercise with either a standard (Sed+Standard, Ex+Standard) or high-protein (Sed+Pro, Ex+Pro) diet. Echocardiography was used to measure cardiac structural remodeling and performance. Skinned cardiac fiber bundles were used to determine the active and passive stress properties, maximum shortening velocity, and calcium sensitivity.[Results]Aerobic training was characterized structurally by increases in ventricle volume (Ex+Standard, 19%; Ex+Pro, 29%) and myocardial thickness (Ex+Standard, 26%; Ex+-Pro, 12%) compared to that of baseline. Skinned trabecula r fiber bundles also had a greater unloaded shortening velocity (Sed+Standard, 1.04±0.05; Sed+Pro, 1.07±0.03; Ex-+Standard, 1.16±0.04; Ex+Pro, 1.18±0.05 FL/s) and calcium sensitivity (pCa₅₀: Sed+Standard, 6.04±0.17; Sed+Pro, 6.08±0.19; Ex+Standard, 6.30±0.09; Ex+Pro, 6.36±0.12) in trained hearts compared to that of hearts from sedentary animals. However, the addition of a high-protein diet did not provide additional benefits to either the structural or mechanical adaptations of the myocardium.[Conclusion]Therefore, it seems that a high-whey-protein diet does not significantly enhance adaptations of the heart to aerobic exercise in comparison to that of a standard diet.     

Cholesterol distribution and structural differentiation in the sarcoplasmic reticulum of rat cardiac muscle cells

Summary The polyene compound, filipin, was used as a probe to localize cholesterol in the membranes of the rat cardiac muscle cell, with particular reference to the sarcoplasmic reticulum (SR). Filipin binds specifically to cholesterol (and related 3--hydroxysterols) in membranes, producing distinct deformations which can be viewed by freeze-fracture and used as markers for the presence of cholesterol-rich regions in the membrane plane. In freeze-fracture replicas of filipin-treated rat myocardium, the muscle cells revealed abundant deformations in their plasma membranes, no deformations in mitochondrial membranes, and an intermediate response in the SR. These results are in agreement with the levels of cholesterol reported in isolated fractions of the different membrane types, and confirm the specificity of filipin action. Within the SR, the filipin-induced deformations were not randomly distributed but occurred more commonly in free SR at or near the Z-region of the sarcomere than in other parts of the free SR or the junctional SR. This finding is interpreted as evidence for a non-homogeneous distribution of cholesterol in cardiac muscle cell SR. The possible significance of cholesterol in relation to structural differentiation and function of the SR is discussed.     

Induction of amino acid transporters expression by endurance exercise in rat skeletal muscle

We here investigated whether an acute bout of endurance exercise would induce the expression of amino acid transporters that regulate leucine transport across plasma and lysosomal membranes in rat skeletal muscle. Rats ran on a motor-driven treadmill at a speed of 28 m/min for 90 min. Immediately after the exercise, we observed that expression of mRNAs encoding l-type amino acid transporter 1 (LAT1) and CD98 was induced in the gastrocnemius, soleus, and extensor digitorum longus (EDL) muscles. Sodium-coupled neutral amino acid transporter 2 (SNAT2) mRNA was also induced by the exercise in those three muscles. Expression of proton-assisted amino acid transporter 1 (PAT1) mRNA was slightly but not significantly induced by a single bout of exercise in soleus and EDL muscles. Exercise-induced mRNA expression of these amino acid transporters appeared to be attenuated by repeated bouts of the exercise. These results suggested that the expression of amino acid transporters for leucine may be induced in response to an increase in the requirement for this amino acid in the cells of working skeletal muscles.     

有氧运动训练影响大鼠胸主动脉环内皮依赖性舒张功能机制的研究

目的:探讨有氧运动对大鼠胸主动脉内皮依赖性舒张功能影响的机制。方法:12只SD大鼠随机分为有氧运动和对照两组(n=6),经过8周每天1 h的游泳训练后(每周5天),测定比较2组间大鼠胸主动脉舒张功能的改变。结果:有氧运动组一氧化氮(NO)和前列环素PGI2途径胸主动脉舒张功能的Rmax值较对照组明显提高(P<0.05)。结论:有氧运动对大鼠胸主动脉舒张功能的有益影响,主要是由NO和PGI2途径介导的。     

Effects of long-term resistance exercise training on autophagy in rat skeletal muscle of chloroquine-induced sporadic inclusion body myositis

Purpose

We examined whether resistance exercise training restores impaired autophagy functions caused by Chloroquine (CQ)-induced Sporadic Inclusion Body Myositis (sIBM) in rat skeletal muscle.

Methods

Male wistar rats were randomly assigned into three groups: Sham (n = 6), CQ (n = 6), and CQ + Exercise (CE, n = 6). To create a rat model of sIBM, rats in the CQ and CE group were intraperitoneally injected with CQ 5 days a week for 16 weeks. Rats in the CE group performed resistance exercise training 3 times a week for 8 weeks in conjunction with CQ starting from week 9 to week 16. During the training period, maximal carrying load, body weight, muscle weight, and relative muscle weight were measured. Autophagy responses were examined by measuring specific markers.

Results

While maximal carrying capacity for resistance exercise training was dramatically increased in the CE group, no significant changes occurred in the skeletal muscle weight as well as in the relative muscle weight of CE compared to the other groups. CQ treatment caused significant increases in the levels of Beclin-1 and p62, and decreases in the levels of LAMP-2 proteins. Interestingly, no significant differences in the LC3-II/I ratio or the LC3-II protein levels were observed. Although CQ-treatment groups suppressed the levels of the potent autophagy inducer, BNIP3, p62 levels were decreased in only the CE group.

Conclusion

Our findings demonstrate that sIBM induced by CQ treatment results in muscle degeneration via impaired autophagy and that resistance exercise training improves movable loading activity. Finally, regular exercise training may provide protection against sIBM by enhancing the autophagy flux through p62 protein.     

补糖和/或刺五加对大鼠运动后骨骼肌细胞AMPK蛋白表达的影响

目的:研究补糖和刺五加对大鼠运动后骨骼肌细胞的AMP激活蛋白激酶(AMPK)蛋白表达的影响及其恢复期的时相性变化。方法:128只SD大鼠大鼠随机分为训练对照组(C组)、训练补糖组(G组)、训练补刺五加皂甙组(A组)和训练补糖补刺五加皂甙组(GA组)四大组,补糖和刺五加均在运动后0.5 h内灌胃给予。根据运动前和运动后不同时间(0 h,4 h,12 h)采样,共分为16小组(n=8)。采用Western blot方法分析骨骼肌的AMPK蛋白含量。结果:①运动后骨骼肌的AMPK蛋白表达量上调,运动后即刻最高(209.23±21.32),随后逐渐恢复;②补药显著地提高了机体在消耗糖原运动后即刻和4 h后的股四头肌AMPK蛋白含量(225.11±20.58和186.31±15.26vs195.19±13.31和157.11±16.43),运动后12 h两组间没有差异;③补糖对骨骼肌AMPK的蛋白表达量的影响没有统计学意义;④补糖同时补药可提高运动后即刻和4 h后的股四头肌AMPK蛋白含量(217.96±19.25和191.86±14.69),但是运动后12h反而低于对照组(121.89±15.23vs137.92±16.01)。结论:运动可激活骨骼肌细胞AMPK,补充刺五加皂甙可上调运动后的AMPK蛋白表达,补糖则没有影响。