Mitochondrial biogenesis in mammals: the role of endogenous nitric oxide

Nitric oxide was found to trigger mitochondrial biogenesis in cells as diverse as brown adipocytes and 3T3-L1, U937, and HeLa cells. This effect of nitric oxide was dependent on guanosine 3',5'-monophosphate (cGMP) and was mediated by the induction of peroxisome proliferator-activated receptor gamma coactivator 1alpha, a master regulator of mitochondrial biogenesis. Moreover, the mitochondrial biogenesis induced by exposure to cold was markedly reduced in brown adipose tissue of endothelial nitric oxide synthase null-mutant (eNOS-/-) mice, which had a reduced metabolic rate and accelerated weight gain as compared to wild-type mice. Thus, a nitric oxide-cGMP-dependent pathway controls mitochondrial biogenesis and body energy balance.     

PGC-1α differentially regulates the mRNA expression profiles of genes related to myofiber type specificity in chicken

Previous studies on mammals showed that peroxisome proliferator-activated receptor gamma coactivator-1α(PGC-1α) played a prominent role in regulating muscle fiber type transition and composition. However, the role of PGC-1α in chicken muscle has seldom been explored. To investigate the effect of PGC-1α on chicken skeletal muscles in this study, the PGC-1α gene was overexpressed or silenced in chicken primary myoblasts by using lentivirus, and then the effects of the PGC-1α gene overexpression and knockdown on the mRNA expression profile of genes related to myofiber type specificity were examined during fiber formation. The results showed that overexpression of PGC-1α from proliferation to differentiation was accompanied by the up-regulated expression of Pax7, MyoD, and CnAα, which was significantly(P0.01) increased after one day of transfection(1 I). The enhancement of MyoG, MEF2 c, and MyHC SM expression lagged, which was improved significantly(P0.01) after four days of transfection(1 I3 D). Overexpression of PGC-1α decreased(P0.01) the MyHC FWM expression after four days of transfection(1 I3 D), and it had no significant impact(P0.05) on the expression of CnB1, NFATc3, and MyHC FRM during myofiber formation. The effective silence(P0.01) of PGC-1α by lentivirus mediating short hairpin RNA(shRNA) was detected after four days of transfection(1 I3 D) in cultures, and the lack of its function in chicken primary myoblasts significantly(P0.01) down-regulated the expression of Pax7, MyoD, CnAα, MyoG, MEF2 c, and MyHC SM, significantly(P0.01) up-regulated the expression of MyHC FWM, and had no significant impact(P0.05) on the expression of CnB1, NFATc3, and MyHC FRM. These results indicated that the role of PGC-1α in regulating the fiber type specificity of chicken skeletal muscles might be similar to that in mammals, which interplayed with key genes related to myocyte differentiation and calcineurin signaling pathway.     

白藜芦醇和EGCG联用对小鼠骨骼肌抗氧化能力的影响

【目的】研究白藜芦醇（Resveratrol,RES）和表没食子儿茶素没食子酸酯（Epigallocatechin-3-Gallate, EGCG）联合使用对小鼠骨骼肌抗氧化能力影响的机制,为开发能通过提高畜禽骨骼肌抗氧化能力进而改善肉品质的新型、绿色、安全的饲料添加剂提供理论依据。【方法】将体重18～22 g的清洁级昆明种小鼠40只,随机分为4组,即二甲基亚砜（DMSO）对照组（CK）、RES组、EGCG组和RES+EGCG联用组,每组10只,采取灌胃的方式对应给药,剂量为每天400 mg/kg,连续给药28 d后测定小鼠骨骼肌中抗氧化酶活力指标,并通过荧光定量PCR方法测定小鼠骨骼肌中抗氧化相关基因的表达量。【结果】RES和EGCG联用小鼠的采食量、体重和脏器指数无显著影响（P>0.05）。与CK相比,RES组、EGCG组及RES+EGCG组血清和肌肉中丙二醛（MDA）含量显著降低（P<0.05,下同）,谷胱甘肽过氧化物酶（GSH-Px）、总超氧化物歧化酶（T-SOD）活力显著升高,小鼠腓肠肌中GSH-Px、超氧化物歧化酶1(SOD1)、超氧化物歧化酶2(SOD2)和核...     

Calorie restriction promotes mitochondrial biogenesis by inducing the expression of eNOS

Calorie restriction extends life span in organisms ranging from yeast to mammals. Here, we report that calorie restriction for either 3 or 12 months induced endothelial nitric oxide synthase (eNOS) expression and 3',5'-cyclic guanosine monophosphate formation in various tissues of male mice. This was accompanied by mitochondrial biogenesis, with increased oxygen consumption and adenosine triphosphate production, and an enhanced expression of sirtuin 1. These effects were strongly attenuated in eNOS null-mutant mice. Thus, nitric oxide plays a fundamental role in the processes induced by calorie restriction and may be involved in the extension of life span in mammals.     

Nebulin and N-WASP cooperate to cause IGF-1-induced sarcomeric actin filament formation

Insulin-like growth factor 1 (IGF-1) induces skeletal muscle maturation and enlargement (hypertrophy). These responses require protein synthesis and myofibril formation (myofibrillogenesis). However, the signaling mechanisms of myofibrillogenesis remain obscure. We found that IGF-1-induced phosphatidylinositol 3-kinase-Akt signaling formed a complex of nebulin and N-WASP at the Z bands of myofibrils by interfering with glycogen synthase kinase-3β in mice. Although N-WASP is known to be an activator of the Arp2/3 complex to form branched actin filaments, the nebulin-N-WASP complex caused actin nucleation for unbranched actin filament formation from the Z bands without the Arp2/3 complex. Furthermore, N-WASP was required for IGF-1-induced muscle hypertrophy. These findings present the mechanisms of IGF-1-induced actin filament formation in myofibrillogenesis required for muscle maturation and hypertrophy and a mechanism of actin nucleation.     

Increased Wnt signaling during aging alters muscle stem cell fate and increases fibrosis

The regenerative potential of skeletal muscle declines with age, and this impairment is associated with an increase in tissue fibrosis. We show that muscle stem cells (satellite cells) from aged mice tend to convert from a myogenic to a fibrogenic lineage as they begin to proliferate and that this conversion is mediated by factors in the systemic environment of the old animals. We also show that this lineage conversion is associated with an activation of the canonical Wnt signaling pathway in aged myogenic progenitors and can be suppressed by Wnt inhibitors. Furthermore, components of serum from aged mice that bind to the Frizzled family of proteins, which are Wnt receptors, may account for the elevated Wnt signaling in aged cells. These results indicate that the Wnt signaling pathway may play a critical role in tissue-specific stem cell aging and an increase in tissue fibrosis with age.     

利用酵母双杂交系统筛选猪Calsarcin-1基因相互作用蛋白

 【目的】利用酵母双杂交系统研究与猪Calsarcin-1相互作用的蛋白,了解Calsarcin-1在猪骨骼肌纤维类型形成和信号通路的调控功能。【方法】首先用长白猪Calsarcin-1基因构建既无自激活性又无毒性的pGBKT7-CS1诱饵载体;然后从猪的骨骼肌组织样中提取mRNA,利用SMART技术合成并纯化双链cDNA;最后通过酵母双杂交系统的共转化法筛选与Calsarcin-1相互作用的蛋白,在GenBank中比对分析相互作用基因,分析Calsarcin-1在骨骼肌中的功能。【结果】构建了pGBKT7-CS1诱饵载体,获得具有完整3′端的猪骨骼肌单链cDNA,并合成双链cDNA。筛选出4个与Calsarcin-1相互作用的蛋白,经与人的基因比较分析,发现Calsarcin-1与α-1肌动蛋白和α-3辅肌动蛋白互作,与骨骼肌Z线附近结构形成有关;与肌集钙蛋白-1和肌钙蛋白T3互作,影响钙离子的调控。【结论】分析所筛选出的蛋白功能,推测Calsarcin-1通过结合这些蛋白,维持细胞结构的稳定,影响相关蛋白的钙离子结合能力,从而参与钙离子调控;而钙离子浓度的变化将影响到其它控制肌纤维分化的因子,或其它调控通路,共同调节快慢肌纤维的形成与转化。     

有氧运动对小鼠心肌、骨骼肌线粒体SOD、MDA、LDH的影响

探讨了有氧运动对小鼠心肌、骨骼肌线粒体SOD、MDA、LDH的影响.结果表明,在3 h游泳运动后,试验组与对照组小鼠心肌中SOD的活性无显著差异(P>0.05),而骨骼肌中SOD活性显著降低(P<0.05);试验组小鼠心肌、骨骼肌线粒体中MDA的含量均有所升高,差异显著(P<0.05):LDH活性有所降低,但差异不显著(P>0.05).说明有氧运动对小鼠骨骼肌中SOD活性和小鼠心肌、骨骼肌线粒体中MDA含量的影响显著,但对LDH活性无显著影响.     

基于绵羊胚胎骨骼肌蛋白质组学的PI3K-AKT信号通路分析

【目的】绵羊是重要的经济动物,其骨骼肌生长发育与产肉性能密切相关。胚胎期是绵羊骨骼肌生长发育的关键阶段,挖掘分析绵羊胚胎骨骼肌蛋白质组数据,为揭示绵羊肌肉发育重要时间节点、筛选绵羊胚胎骨骼肌生长发育调控蛋白质提供依据。【方法】本团队已对妊娠第85天、第105天和第135天的中国美利奴绵羊胚胎背最长肌进行串联质谱(tandem mass tag, TMT)蛋白质定量,鉴定到1316种差异丰度蛋白质。现利用GO、KEGG和R等方法对这些差异丰度蛋白质开展聚类、功能注释和通路分析等生物信息学分析。【结果】基于前期研究结果对差异丰度蛋白质进行R语言聚类,分析结果显示,cluster 5类蛋白在胚胎骨骼肌第105天具有较高丰度。对cluster 5 蛋白进行GO和KEGG富集分析发现,该类蛋白质参与胞内蛋白质代谢过程,显著富集于PI3K-AKT信号通路中,而在该信号通路中RAC-β丝氨酸/苏氨酸蛋白激酶X1(AKT2)具有较高表达丰度。蛋白质生物信息学结果表明,AKT2蛋白由481个氨基酸构成,AKT2蛋白理论分子量为55.58kD,由66个带正电荷的氨基酸残基和72个带负电荷的氨基酸残基组成,理论等电点为6.08,亲水性平均系数-0.454,属于亲水性蛋白。预测AKT2蛋白的481个氨基酸全部位于膜外,属于膜受体蛋白。AKT2蛋白有12个N-端糖基化位点,71个磷酸化位点,与蛋白酶K相似度为99%,属于蛋白酶催化亚基家族。【结论】绵羊胚胎骨骼肌蛋白质组数据发现,第105天是绵羊胚胎骨骼肌纤维由增殖分化到增大增粗的转折点,具有调控绵羊胚胎骨骼肌纤维生长发育作用的PI3K-AKT信号通路在该节点显著富集,AKT2是调控该信号通路的重要候选蛋白。综上,本研究结果对揭示胚胎骨骼肌生长发育及其调控分子机制具有重要理论指导意义。     

虾青素对大强度运动致大鼠骨骼肌氧化应激损伤及细胞凋亡的影响

目的研究人参皂苷CK (ginsenoside compound K) 对力竭游泳大鼠抗疲劳作用及骨骼肌氧化应激的影响。方法将SD大鼠分为安静对照组、运动模型组及人参皂苷CK低、中、高剂量组 (10、20和40 mg/kg),安静对照组及运动模型组用等体积的蒸馏水灌胃,每天1次,连续4周;安静对照组大鼠不参加运动,其余各组大鼠于给药当天进行游泳训练。在末次给药结束后,进行力竭游泳试验,记录大鼠力竭游泳时间,检测血清抗疲劳指标[ 睾酮 (TTS)、皮质酮 (CTC)、乳酸 (LA)和尿素氮 (BUN)]、肌糖原和肝糖原储备水平、骨骼肌病理改变、骨骼肌氧化应激指标[ 过氧化氢酶 (CAT)、谷胱甘肽过氧化物酶 (GSH-Px)、超氧化物歧化酶 (SOD)和丙二醛 (MDA)]及Nrf2和HO-1蛋白表达。结果与运动模型组相比,人参皂苷CK低、中、高剂量组大鼠力竭游泳时间、血清TTS水平、肌糖原和肝糖原储备水平、骨骼肌CAT、GSH-Px和SOD活性及Nrf2和HO-1蛋白表达增加 (P<0.05或P<0.01),骨骼肌病理改变减轻,血清CTC、LA和BUN水平以及骨骼肌MDA水平降低 (P<0.05或P<0.01)。结论人参皂苷CK具有减轻力竭游泳大鼠疲劳的作用,该作用与激活Nrf2/HO-1信号通路有关,进而增强骨骼肌的抗氧化应激能力。     

褪黑素和烟酰胺单核苷酸对鹅骨骼肌卫星细胞增殖的影响

【背景】目前关于褪黑素（melatonin,MLT）的研究多集中在家禽的生殖功能上,而与家禽肌肉发育相关的作用机制却鲜有研究。同时MLT与烟酰胺单核苷酸(nicotinamide mononucleotide, NMN)功能相似且都与昼夜节律相关,研究发现MLT与NMN的单一处理对细胞衰老的影响有限,而共同处理的效果更为显著,虽然二者对线粒体功能和骨骼肌衰老的影响也有相关报道,但在骨骼肌生长发育的作用机制方面尚无可以参考的报道。【目的】通过探究MLT与NMN参与浙东白鹅骨骼肌卫星细胞增殖的分子机制,为其在家禽生产实践中的应用提供思路。【方法】通过解剖鹅胚分离培养鹅骨骼肌卫星细胞,并对骨骼肌卫星细胞的特异性蛋白Pax7和Desmin进行免疫荧光染色以此鉴定细胞,在体外成熟培养基础上用1 ng·mL-1 MLT与1μg·mL-1 NMN分别单独或组合处理细胞24 h后,利用CCK-8检测细胞活力;为探究MLT如何调控鹅骨骼肌卫星细胞增殖的机制,通过构建MLT受体基因（MTNR1A、MTNR1B）的过表达载体,并与1 ng·mL-1 MLT共同处理细胞,采用qRT-PCR和Western b...     

常见生物模型抗氧化活性评价方法的研究进展

目的研究黄秋葵多糖对耐力训练小鼠自由基代谢和糖代谢的影响。方法选取昆明种健康雄性小鼠为试验对象,以负荷游泳训练方式建立耐力运动模型。将试验小鼠随机分为安静组 (A组) 、对照组(B组)和黄秋葵多糖组 (低、中、高剂量组分别为C₁、C₂、C₃组) 进行试验小鼠活动状态行为学观察和体质量测定,并测定肌酸激酶 (CK)、血清乳酸脱氢酶 (LDH)、血乳酸 (BL)、血尿素氮 (BUN)、谷丙转氨酶 (GPT)和谷草转氨酶 (GOT)、肌糖原和肝糖原含量以及组织抗氧化指标超氧化物歧化酶(SOD)活性、过氧化氢酶 (CAT) 活性和丙二醛 (MDA) 含量。结果与A组相比,B组体质量增长缓慢,小鼠疲劳特征明显,游泳至力竭时间短,CK、LDH、BL、BUN、GPT和GOT含量升高,肌糖原和肝糖原含量无显著变化,肝脏SOD活性升高,肝脏和骨骼肌CAT活性降低,肝脏和骨骼肌MDA含量升高。C₃组 (400 mg/kg剂量) 黄秋葵多糖耐力训练小鼠体质量增加,行为学特征改善,游泳至力竭时间延长,CK、LDH、BL、BUN、GPT和GOT含量降低,肌糖原和肝糖原含量升高,肝脏SOD活性降低,肝脏和骨骼肌CAT活性升高,肝脏和骨骼肌MDA含量降低。结论黄秋葵多糖有助于改善耐力训练小鼠自由基代谢和糖代谢,从而提高小鼠运动能力,发挥抗疲劳功能。     

Role of adenine nucleotide translocator 1 in mtDNA maintenance

Autosomal dominant progressive external ophthalmoplegia is a rare human disease that shows a Mendelian inheritance pattern, but is characterized by large-scale mitochondrial DNA (mtDNA) deletions. We have identified two heterozygous missense mutations in the nuclear gene encoding the heart/skeletal muscle isoform of the adenine nucleotide translocator (ANT1) in five families and one sporadic patient. The familial mutation substitutes a proline for a highly conserved alanine at position 114 in the ANT1 protein. The analogous mutation in yeast caused a respiratory defect. These results indicate that ANT has a role in mtDNA maintenance and that a mitochondrial disease can be caused by a dominant mechanism.     

阻断PI3K/AKT通路通过激活FoxO1抑制 猪骨骼肌卫星细胞分化

【目的】在骨骼肌生长或损伤刺激下,骨骼肌卫星细胞被激活、增殖分化形成肌管,促进骨骼肌的生长发育或修复组织创伤。FoxO1负调控骨骼肌的生成,但在骨骼肌卫星细胞分化过程中的作用未见报道。因此,笔者探索FoxO1对猪骨骼肌卫星细胞分化的影响,希望为深入研究FoxO1调控骨骼肌生长发育的作用机理奠定基础。【方法】以1-3日龄健康大白猪为材料,采用单根肌纤维法分离培养猪骨骼肌卫星细胞,接种第2天、第4天和第6天在倒置显微镜下观察细胞形态并拍照。在细胞分化第8天,用免疫荧光染色方法染肌管,DAPI染核,并在荧光倒置显微镜下观察拍照。 待细胞汇合至70%-80%时,将培养基换成含50 nmol•L-1 渥曼青霉素（wortmannin,WM）的分化培养基,分别于细胞分化第0天、第4天和第8 天收集细胞,提取总RNA和总蛋白,采用real-time qPCR和Western blotting方法检测WM对FoxO1以及骨骼肌卫星细胞分化标志基因表达的影响。【结果】猪骨骼肌卫星细胞在接种第2天开始贴壁,呈梭形。第4天细胞数量增加,部分发生融合。第6天时细胞呈方向性生长。第8天细胞进一步融合形成肌管。WM处理组的FoxO1 mRNA表达水平未发生显著变化（P＞0.05）,非磷酸化的FoxO1蛋白表达显著高于对照组（P＜0.05）,而p-FoxO1蛋白表达较对照组显著下降（P＜0.05）。WM处理组的细胞在分化第8天,虽然也出现了蜂窝状生长,但是与对照组相比细胞未呈方向性生长并形成肌管。Western blotting结果显示,WM明显抑制猪骨骼肌卫星细胞分化早期标志基因MyoD、中后期标志基因MyoG和末期标志基因MyHC蛋白的表达。【结论】以WM阻断PI3K信号通路能使FoxO1去磷酸化,抑制猪骨骼肌卫星细胞的分化,延迟肌管的形成,并降低成肌分化标志基因MyoD、MyoG和MyHC的表达。总之,阻断PI3K信号通路通过激活FoxO1抑制猪骨骼肌卫星细胞分化。     

TGF-β/Smad信号通路在Follistatin调节鸭骨骼肌卫星细胞增殖过程中的作用机制

【目的】卵泡抑素（Follistatin）能够调节骨骼肌肥大和脂肪沉积,可促进骨骼肌卫星细胞增殖。拟采用体外重组 Follistatin处理增殖期的鸭骨骼肌卫星细胞,阐明TGF-β/Smad信号通路在Follistatin调节鸭骨骼肌卫星细胞增殖过程中的作用机制。【方法】以孵化14 d的鸭胚为试验材料,采用差速贴壁的方法分离骨骼肌卫星细胞,待细胞长到70%-80%时,将培养基换成含有浓度分别为0、1、10、100 ng·mL^-1的Follistatin培养基,继续培养36 h后,采用CCK-8检测骨骼肌卫星细胞增殖情况;使用抗pax7抗体染色,DAPI染核,鉴定骨骼肌卫星细胞;采用real-time qPCR方法检测Follistatin对骨骼肌卫星细胞增殖过程中的标记基因PCNA、生肌因子基因MyoD和TGF-β信号通路中TGF-β、Smad2和Smad3的表达的影响。【结果】在倒置显微镜下观察,传代培养12 h鸭骨骼肌细胞一部分未贴壁呈圆形,一部分贴壁呈梭形。24 h后细胞全部贴壁,细胞略有变长。2 d后细胞继续增多,且呈长梭形。3 d后细胞数目增加,个别细胞融合。4 d后细胞数目进一步增加,细胞变粗,个别细胞融合。5 d后有少量细胞开始分化,细胞进一步融合。Pax7免疫荧光染色分析显示,95%以上的细胞中Pax7呈阳性表达;CCK-8检测细胞增殖分析表明,不同浓度的Follistatin处理鸭骨骼肌卫星细胞后,各处理组细胞增殖均显著高于对照组(P＜0.01),且10 ng·mL^-1 Follistatin处理鸭骨骼肌卫星细胞增殖效果最明显,为最佳处理浓度;与对照组相比,10 ng·mL^-1 Follistatin处理组的MyoD基因表达量显著下降(P＜0.05),PCNA基因表达量显著升高(P＜0.05),Myf5基因表达量显著升高,TGF-β和Smad2基因表达显著升高(P＜0.05),且Smad3基因表达量极限著升高(P＜0.01);Western blotting检测蛋白表达水平结果表明,与对照组相比,TGF-β、Smad2 和Smad3磷酸化水平也显著升高。【结论】10 ng·mL^-1 Follistatin能显著促进鸭骨骼肌卫星细胞增殖,这一过程可通过TGF-β/Smad信号通路实现。使用最佳Follistatin处理浓度能够显著促进鸭骨骼肌卫星细胞增殖,该研究为鸭骨骼肌生长发育调控机理研究奠定分子基础。     

藏雪莲水提取物对小白鼠组织中一氧化氮含量的影响

[目的]研究藏雪莲水提取物对小白鼠组织中一氧化氮含量的影响,为藏雪莲抗缺氧机制的研究提供参考。[方法]将100只小白鼠随机分为高剂量组、中剂量组、低剂量组和对照组,连续24 d分别灌服不同剂量的藏雪莲水提取物和生理盐水,然后处死小白鼠快速采取肝、肺、心肌和骨骼肌,测定各组织内一氧化氮的含量。[结果]结果表明,高剂量组的肝、肺和骨骼肌和中剂量组的肝中一氧化氮含量中显著高于对照组和低剂量组,差异极显著(P<0.01);中剂量组的肺和骨骼肌中一氧化氮含量高于对照和低剂量组,差异显著(P<0.05);对照组的心肌中一氧化氮含量高于各药物组,差异极显著(P<0.01)。[结论]藏雪莲能够提高小白鼠肝、肺和骨骼肌中一氧化氮的含量,降低心肌中的含量。