八眉猪、长白猪及长×八杂交猪肌肉组织中FoxO1基因的表达

分别取6月龄八眉、长白和 (长×八)杂交猪后腿部比目鱼肌、腓肠肌和趾长伸肌, 提取总RNA, 根据人、黑猩猩及大鼠等物种FoxO1基因同源序列设计并合成引物, 以猪b-actin 基因作为内参, 优化反应条件和体系, RT-PCR 单管扩增猪FoxO1基因, 检测八眉、长白和长×八杂交猪不同类型骨骼肌中FoxO1基因mRNA的表达差异。结果表明: 在不同经济类型猪群和不同类型骨骼肌中FoxO1基因mRNA的表达丰度不同, 即在八眉 猪骨骼肌中的表达普遍高于长白猪 (P<0.01), 在杂交组合 (长×八)骨骼肌中的表达也高于长白猪 (P<0.01); 同时在以Ⅰ型纤维为主的比目鱼肌中表达丰度最低(P<0.01), 在以Ⅱb型纤维为主的趾长伸肌中表达丰度最高(P<0.01)。结果提示, FoxO1基因的表达与Ⅰ型肌纤维的含量成反比; 不同经济类型猪品种骨骼肌的发育与FoxO1基因的调控有关。     

Cloning and characterization of a calmodulin gene (CaM) in crayfish Procambarus clarkii and expression during molting

Calmodulin (CaM) is a highly conserved calcium (Ca²⁺) binding protein that transduces Ca²⁺ signals into downstream effects influencing a range of cellular processes, including Ca²⁺ homeostasis. The present study explores CaM expression when Ca²⁺ homeostasis is challenged during the mineralization cycle of the freshwater crayfish (Procambarus clarkii). In this paper we report the cloning of a CaM gene from axial abdominal crayfish muscle (referred to as pcCaM). The pcCaM mRNA is ubiquitously expressed but is far more abundant in excitable tissue (muscle, nerve) than in any epithelia (gill, antennal gland, digestive) suggesting that it plays a greater role in the biology of excitation than in epithelial ion transport. In muscle cells the pcCaM was colocalized on the plasma membrane with the Ca²⁺ ATPase (PMCA) known to regulate intracellular Ca²⁺ through basolateral efflux. While PMCA exhibits a greater upregulation in epithelia (than in non-epithelial tissues) during molting stages requiring transcellular Ca²⁺ flux (pre- and postmolt compared with intermolt), expression of pcCaM exhibited a uniform increase in epithelial and non-epithelial tissues alike. The common increase in expression of CaM in all tissues during pre- and postmolt stages (compared with intermolt) suggests that the upregulation is systemically (hormonally) mediated. Colocalization of CaM with PMCA confirms physiological findings that their regulation is linked.     

Hyperinsulinemia and ectopic fat deposition can develop in the face of hyperadiponectinemia in young obese rats

Serum adiponectin has been reported to inversely correlate with the degree of adiposity in children. However, the relative contribution of adiponectin-dependent signaling to the development of metabolic syndrome in childhood obesity is unclear. We overfed prepubertal, male Sprague-Dawley rats a high-fat diet via total enteral nutrition. Excessive caloric intake led to obesity, increased body weight and fat mass; dyslipidemia; ectopic fat deposition; and hyperinsulinemia (P<.05). Expression of fatty acid transporter FAT/CD36 was elevated in both liver and skeletal muscle (P<.05). Hepatic Akt phosphorylation was elevated (P<.05) and FoxO1 protein in hepatic nuclear extracts was reduced (P<.05) in the face of hyperinsulinemia, whereas no increase in Akt phosphorylation or decrease in nuclear FoxO1 was observed in skeletal muscle. Overfeeding increased serum adiponectin concentration from 24.6±1.9 μg/ml to 46.3±5.9 μg/ml (P<.004), and positively correlated with increased adipose tissue mass. The expression of the inflammatory cytokine tumor necrosis factor α in the adipose tissue was unchanged. Adiponectin-mediated adenosine monophosphate (AMP) kinase phosphorylation, peroxisome proliferator-activator receptor-α expression and the expression of genes involved in fatty acid oxidation were elevated in both liver and muscle (P<.05). These data (1) demonstrate that excessive intake of a high-fat diet in young rats results in “adiponectin-independent” increases in ectopic fat deposition and hyperinsulinemia, (2) suggest that fatty acid transport is a major mechanism underlying ectopic fat deposition, (3) demonstrate tissue-specific differences in the response of Akt-FoxO signaling to hyperinsulinemia following the development of pediatric obesity and (4) suggest age-related differences in the role of adiponectin in pathological responses associated with obesity.     

Molecular characterization of the sarcoplasmic calcium-binding protein (SCP) from crayfish Procambarus clarkii

Sarcoplasmic Calcium-binding Protein (SCP) is believed to function as the invertebrate equivalent of vertebrate parvalbumin, namely to “buffer” cytosolic Ca²⁺. We have cloned and characterized a novel SCP from axial abdominal muscle of crayfish Procambarus clarkii (referred to as pcSCP1), and have examined tissue specific distribution and expression as a function of molting stage in non-epithelial and epithelial tissues. The complete sequence of pcSCP1 consists of 1052 bp with a 579 bp open reading frame, coding for 193 amino acid residues (molecular mass of 21.8 kDa). There is a 387 bp 3′ terminal non-coding region with a poly (A) tail. The deduced pcSCP1 protein sequence matched most closely with published SCP sequences from another crayfish Astacus leptodactylus (92.8%) and from shrimp (78.6–81.2%) and fruit fly (53%). Real-time PCR analysis confirmed that pcSCP1 is ubiquitously expressed in all tissues tested (gill, hepatopancreas, intestine, antennal gland, muscle); however it is most abundant in muscle particularly in the axial abdominal muscle. The real-time PCR analysis revealed that pcSCP1 expression is downregulated in pre- and postmolt stages compared with intermolt. Epithelial (hepatopancreas and antennal gland) SCP expression exhibited a more dramatic decrease than that observed in muscle. Expression trends for pcSCP1 paralleled published trends for sarco/endoplasmic reticular calcium ATPase (SERCA), suggesting that their cellular function in regulating intracellular Ca²⁺ is linked.     

FoxO1抑制猪骨骼肌MyHC Ⅰ的表达

为研究FoxO1与骨骼肌纤维类型之间的关系,本试验以大白猪为实验材料,利用RT-PCR和Western印迹技术,检测了FoxO1与肌纤维类型标志基因MyHCⅠ、MyHCⅡa、MyHCⅡb和MyHCⅡx在特定骨骼肌中的表达规律,以及调控肌纤维类型关键基因Mef2c和NFAT的表达,并用Wortmannin处理原代培养的猪骨骼肌成肌细胞,检测了FoxO1与肌纤维类型相关基因的表达.结果显示,FoxO1在不同骨骼肌类型中mRNA表达差异不显著(P0.05),其蛋白表达与MyHC各亚型显著相关.Wortmannin处理结果显示,在处理的第3和5d,FoxO1蛋白与MyHCⅡb,MyHCⅡx和NFAT表达显著正相关,而与MyHCⅠ,MyHCⅡa和Mef2c表达显著负相关.结果表明,FoxO1通过抑制MyHCⅠ的表达调控肌纤维类型.     

High CO2 Levels Cause Skeletal Muscle Atrophy via AMP-activated Kinase (AMPK), FoxO3a Protein,and Muscle-specific Ring Finger Protein 1 (MuRF1)

Patients with chronic obstructive pulmonary disease, acute lung injury, and critical care illness may develop hypercapnia. Many of these patients often have muscle dysfunction which increases morbidity and impairs their quality of life. Here, we investigated whether hypercapnia leads to skeletal muscle atrophy. Mice exposed to high CO₂ had decreased skeletal muscle wet weight, fiber diameter, and strength. Cultured myotubes exposed to high CO₂ had reduced fiber diameter, protein/DNA ratios, and anabolic capacity. High CO₂ induced the expression of MuRF1 in vivo and in vitro, whereas MuRF1^−/− mice exposed to high CO₂ did not develop muscle atrophy. AMP-activated kinase (AMPK), a metabolic sensor, was activated in myotubes exposed to high CO₂, and loss-of-function studies showed that the AMPKα2 isoform is necessary for muscle-specific ring finger protein 1 (MuRF1) up-regulation and myofiber size reduction. High CO₂ induced AMPKα2 activation, triggering the phosphorylation and nuclear translocation of FoxO3a, and leading to an increase in MuRF1 expression and myotube atrophy. Accordingly, we provide evidence that high CO₂ activates skeletal muscle atrophy via AMPKα2-FoxO3a-MuRF1, which is of biological and potentially clinical significance in patients with lung diseases and hypercapnia.     

抗阻训练通过Akt-FoxO1通路抑制低氧诱导的骨骼肌萎缩

高原低氧环境会引起肌力下降和运动能力退化,而抗阻训练是刺激骨骼肌生长的重要手段,叉头转录因子1（fork head box protein O 1,FoxO1）在调控骨骼肌蛋白质分解通路中承担重要角色。为探究Akt-FoxO1通路是否参与抗阻训练抑制低氧诱导的骨骼肌萎缩,本研究构建低氧诱导骨骼肌萎缩的大鼠模型,并模拟海拔4 000 m低氧环境下（12.4% O2）进行抗阻训练,对比观察大鼠比目鱼肌和趾长伸肌湿重和横截面积,以及蛋白激酶B（protein kinase B,Akt）、叉头转录因子1、泛素蛋白连接酶1（muscle ring finger 1,MuRF1）的表达差异等。结果表明,低氧暴露导致大鼠趾长伸肌湿重显著下降,苏木精-伊红染色组织切片分析肌纤维横截面积、低氧环境下比目鱼肌横截面积明显下降,而低氧抗阻训练后趾长伸肌横截面积明显高于安静组。实时荧光定量PCR和蛋白质免疫印迹结果显示,低氧暴露后FoxO1和MuRF1基因表达明显上调,低氧下抗阻训练后发现,Akt基因表达明显上调而FoxO1、MuRF则明显下调。免疫荧光观察磷酸化FoxO1在细胞核内外表达情况,发现抗阻训练后FoxO1（S256）于细胞核外表达增强。上述结果表明,抗阻训练可以达到抑制低氧诱导骨骼肌萎缩的效果,Akt促进FoxO1磷酸化从而减缓骨骼肌蛋白质分解过程是抗阻训练能够抑制骨骼肌萎缩的分子机制之一。     

Genomic cloning of the Hsc71 gene in the hermaphroditic teleost Rivulus marmoratus and analysis of its expression in skeletal muscle: identification of a novel muscle-preferred regulatory element

To further our understanding of the role of stress proteins in development as well as in adaptation of fish to adverse environmental conditions, we undertook molecular analyses of stress protein encoding genes from the hermaphroditic teleost Rivulus marmoratus. We isolated a genomic clone containing the Hsc71 gene (rm-hsc71m) and its upstream sequences. rm-Hsc71m is not induced by external stress, but is enriched in a tissue-specific manner during early development. In adult, the strongest expression appeared in skeletal muscle, whereas lower expression was seen in the gill, eye and brain. To understand the regulatory basis of high muscle expression of rm-hsc71m, transfection of R.marmoratus muscle tissue was performed using 5′ deletion fragments containing the rm-hsc71m promoter driving EGFP expression. An upstream region from –2.7 to –1.9 kb was identified as a muscle-specific regulatory region. Within this region, we identified at least three sites with the novel sequence TGTnACA interacting with a fish muscle factor having an M_r of 32 000. Our data indicate that rm-hsc71m expression in skeletal muscle is controlled by a muscle-specific regulatory element containing this novel motif.