Chemerin--a new adipokine that modulates adipogenesis via its own receptor

Chemerin, an 18 kDa protein secreted by adipose tissue, was reported to modulate immune system function through its binding to the chemerin receptor (chemerinR). We herein demonstrate that chemerin also influences adipose cell function. Our data showed that chemerin and chemerinR mRNA expressions were highly expressed in adipose tissues, and that their expression levels were up-regulated in mice fed a high-fat diet. Both chemerin and chemerinR mRNA expression dramatically increased during the differentiation of 3T3-L1 cells and human preadipocytes into adipocytes. Furthermore, recombinant chemerin induced the phosphorylation of extracellular signal-regulated kinases 1/2 (ERK 1/2) and lipolysis in differentiated 3T3-L1 adipocytes. Thus, the adipokine chemerin likely regulates adipocyte function by autocrine/paracrine mechanisms.     

Expression profiling of the gamma-subunit isoforms of AMP-activated protein kinase suggests a major role for gamma3 in white skeletal muscle

Expression patterns of the three isoforms of the regulatory gamma-subunit of AMP-activated protein kinase (AMPK) were determined in various tissues from adult humans, mice, and rats, as well as in human primary muscle cells. Real-time PCR-based quantification of mRNA showed similar expression patterns in the three species and a good correlation with protein expression in mice and rats. The gamma3-isoform appeared highly specific to skeletal muscle, whereas gamma1 and gamma2 showed broad tissue distributions. Moreover, the proportion of white, type IIb fibers in the mouse and rat muscle samples, as indicated by real-time PCR quantification of Atp1b2 mRNA, showed a strong positive correlation with the expression of gamma3. In samples of white skeletal muscle, gamma3 clearly appeared to be the most abundant gamma-isoform. Differentiation of human primary muscle cells from myoblasts into multinucleated myotubes was accompanied by upregulation of gamma3 mRNA expression, whereas levels of gamma1 and gamma2 remained largely unchanged. However, even in these cultured myotubes, gamma2 was the most highly expressed isoform, indicating a considerable difference compared with adult skeletal muscle. Immunoblot analysis of mouse gastrocnemius and quadriceps muscle extracts precipitated with a gamma3-specific antibody showed that gamma3 was exclusively associated with the alpha2- and beta2-subunit isoforms. The observation that the AMPKgamma3 isoform is expressed primarily in white skeletal muscle, in which it is the predominant gamma-isoform, strongly suggests that gamma3 has a key role in this tissue.     

Molecular characterization of the BTG2 and BTG3 genes in fetal muscle development of pigs

BTG2 and BTG3 are two members of the B-cell translocation gene family with anti-proliferative properties. BTG1 gene in this gene family has been reported to play a key role in muscle growth. In this study, we identified and characterized the porcine BTG2 and BTG3 genes, mapped the two genes to porcine chromosomes, and analyzed their expression differences in the longissimus dorci muscle of 33 dpc (day postconception), 65 dpc and 90 dpc in the lean Landrace and fatty Chinese Tongcheng pig breeds. Expression changes in differentiated C2C12 cells were also investigated with myogenin as internal control. The results showed that the porcine BTG2 and BTG3 genes were mapped on SSC9q21-25 and SSC13q47, respectively. BTG2 gene expressed at high levels in skeletal muscle and heart in both Tongcheng and Landrace pigs whereas BTG3 gene expressed at lower levels in skeletal muscle and heart than in other tissues. Furthermore, BTG3 expressed at higher levels in skeletal muscle of Tonghceng compared with Landrace pig. The expression of BTG2 and BTG3 was significantly different in skeletal muscle among different developmental stages and between the two breeds. Expression analysis in murine myoblast cells showed that both genes were induced in differentiated C2C12 cells, suggesting a role of them in myogenic differentiation. Our study indicated that BTG2 and BTG3, especially BTG3 gene, may be important genes for skeletal muscle growth.     

Sarcosin (Krp1) in skeletal muscle differentiation: gene expression profiling and knockdown experiments

SARCOSIN, also named Krp1, has been identified as a protein exclusively expressed in striated muscle tissue. Here we report on the role of SARCOSIN in skeletal muscle development and differentiation. We demonstrate, by means of whole-mount in situ hybridization, that Sarcosin mRNA is expressed in the myotome part of the mature somites in mouse embryos from embryonic day 9.5 onwards. Sarcosin is not expressed in the developing heart at these embryonic stages, and in adult tissues the mRNA expression levels are five times lower in the heart than in skeletal muscle. SARCOSIN protein partially co-localizes with the M-band protein myomesin and between and below laterally fusing myofibrils in adult skeletal muscle tissue. RNA interference mediated knock-down of SARCOSIN in the C2C12 myoblast cell line appeared to be stimulatory in the early phase of differentiation, but inhibitory at a later phase of differentiation.     

Molecular characterization and expression patterns of the actinin-associated LIM protein (ALP) subfamily genes in porcine skeletal muscle

The actinin-associated LIM protein (ALP) subfamily has important functions in cell signal transduction, cell proliferation, and integration of cytoskeletal architecture. To detect their functions in pig skeletal muscle, we cloned and characterized the pig ALP subfamily genes, drew their genomic structure maps, and detected their tissue expression patterns. We identified a new spliced variant of PDLIM3 in pig skeletal muscle and named it as PDLIM3-4, which was only expressed in the heart and skeletal muscle. Our results showed that PDLIM3-4 was expressed in adult pig skeletal muscle with the highest expression level, and both PDLIM3-4 isoform and PDLIM4 had different expression profiles during the prenatal and postnatal stages of skeletal muscle development among the three pig breeds. These studies provide useful information for further research on the functions of pig ALP subfamily genes in skeletal muscle development.     

Syndecan-3 and syndecan-4 specifically mark skeletal muscle satellite cells and are implicated in satellite cell maintenance and muscle regeneration.

Myogenesis in the embryo and the adult mammal consists of a highly organized and regulated sequence of cellular processes to form or repair muscle tissue that include cell proliferation, migration, and differentiation. Data from cell culture and in vivo experiments implicate both FGFs and HGF as critical regulators of these processes. Both factors require heparan sulfate glycosaminoglycans for signaling from their respective receptors. Since syndecans, a family of cell-surface transmembrane heparan sulfate proteoglycans (HSPGs) are implicated in FGF signaling and skeletal muscle differentiation, we examined the expression of syndecans 1-4 in embryonic, fetal, postnatal, and adult muscle tissue, as well as on primary adult muscle fiber cultures. We show that syndecan-1, -3, and -4 are expressed in developing skeletal muscle tissue and that syndecan-3 and -4 expression is highly restricted in adult skeletal muscle to cells retaining myogenic capacity. These two HSPGs appear to be expressed exclusively and universally on quiescent adult satellite cells in adult skeletal muscle tissue, suggesting a role for HSPGs in satellite cell maintenance or activation. Once activated, all satellite cells maintain expression of syndecan-3 and syndecan-4 for at least 96 h, also implicating these HSPGs in muscle regeneration. Inhibition of HSPG sulfation by treatment of intact myofibers with chlorate results in delayed proliferation and altered MyoD expression, demonstrating that heparan sulfate is required for proper progression of the early satellite cell myogenic program. These data suggest that, in addition to providing potentially useful new markers for satellite cells, syndecan-3 and syndecan-4 may play important regulatory roles in satellite cell maintenance, activation, proliferation, and differentiation during skeletal muscle regeneration.     

VITO-1, a novel vestigial related protein is predominantly expressed in the skeletal muscle lineage

In order to identify novel genes expressed in skeletal muscle we performed a subtractive hybridization for genes expressed in human skeletal muscle but not in other tissues. We identified a novel scalloped interaction domain (SID) containing protein in humans and in the mouse, which we named VITO-1. Highest homology of VITO-1 was found with the Drosophila vestigial and the human TONDU proteins in the SID (54 and 40%, respectively). Using whole-mount hybridzation and Northern blot analysis, we showed that VITO-1 is expressed in the somitic myotome from E8.75 mouse embryos onwards and later on in skeletal muscle but not in the heart. Additional expression domains during development were detected in the pharyngeal pouches and clefts starting at E8.0 as well as in the cranial pharynx and in Rathkes pouch. By Northern blot analysis, we found VITO-1 to be up-regulated in C2C12 myotubes although some expression can be detected in proliferating C2C12 myoblasts. No expression was spotted in other adult mouse tissues. Likewise, expression of human Vito-1 during fetal and adult human development was found exclusively in skeletal muscle preferentially in fast skeletal muscles. These data suggest a role of VITO-1 for the development of skeletal muscles and of pharyngeal clefts/Rathkes' pouch derived structures.     

翘嘴鳜miR-222的表达特征

为了解翘嘴鳜miR-222的时空表达规律, 研究利用实时荧光定量PCR的方法检测miR-222在翘嘴鳜不同组织、胚胎发育及胚后发育中的相对表达丰度。研究结果显示, miR-222在肌肉相关的组织中表达较高, 特别是在成年翘嘴鳜的白肌中表达最高; 胚胎发育阶段结果显示, miR-222在胚胎发育的2细胞期就有表达, 而表达量在心动期达到最高。不同组织及不同发育阶段的差异性表达结果表明, miR-222很可能参与调控鳜鱼肌肉的生长发育。为研究合成代谢过程中miR-222在肌肉生长调控中的表达规律, 通过对翘嘴鳜幼鱼在饥饿一周后饱食一餐的实验处理下, 利用实时荧光定量的方法测定miR-222在骨骼肌中的相对表达变化。结果显示, miR-222的表达量在恢复喂食后的1h显著上升(P0.05), 表明miR-222很可能是调节鱼类骨骼肌生长过程中, 参与快速应答信号系统的一类miRNA。研究为miR-222在鱼类发育中的调控作用提供一些理论依据。       

VITO-1, a novel vestigial related protein is predominantly expressed in the skeletal muscle lineage

In order to identify novel genes expressed in skeletal muscle we performed a subtractive hybridization for genes expressed in human skeletal muscle but not in other tissues. We identified a novel scalloped interaction domain (SID) containing protein in humans and in the mouse, which we named VITO-1. Highest homology of VITO-1 was found with the Drosophila vestigial and the human TONDU proteins in the SID (54 and 40%, respectively). Using whole-mount hybridzation and Northern blot analysis, we showed that VITO-1 is expressed in the somitic myotome from E8.75 mouse embryos onwards and later on in skeletal muscle but not in the heart. Additional expression domains during development were detected in the pharyngeal pouches and clefts starting at E8.0 as well as in the cranial pharynx and in Rathkes pouch. By Northern blot analysis, we found VITO-1 to be up-regulated in C2C12 myotubes although some expression can be detected in proliferating C2C12 myoblasts. No expression was spotted in other adult mouse tissues. Likewise, expression of human Vito-1 during fetal and adult human development was found exclusively in skeletal muscle preferentially in fast skeletal muscles. These data suggest a role of VITO-1 for the development of skeletal muscles and of pharyngeal clefts/Rathkes' pouch derived structures.     

The C-terminal nonapeptide of mature chemerin activates the chemerin receptor with low nanomolar potency

Chemerin is a novel protein identified as the natural ligand of ChemR23 (chemerinR), a previously orphan G protein-coupled receptor expressed in immature dendritic cells and macrophages. Chemerin is synthesized as a secreted precursor, prochemerin, which is poorly active, but converted into a full agonist of chemerinR by proteolytic removal of the last six amino acids. In the present work, we have synthesized a number of peptides derived from the C-terminal domain of human prochemerin and have investigated their functional properties as agonists or antagonists of human chemerinR. We found that the nonapeptide (149)YFPGQFAFS(157) (chemerin-9), corresponding to the C terminus of processed chemerin, retained most of the activity of the full-size protein, with regard to agonism toward the chemerinR. Extension of this peptide at its N terminus did not increase the activity, whereas further truncations rapidly resulted in inactive compounds. The C-terminal end of the peptide appeared crucial for its activity, as addition of a single amino acid or removal of two amino acids modified the potency by four orders of magnitude. Alanine-scanning mutagenesis identified residues Tyr(149), Phe(150), Gly(152), Phe(154), and Phe(156) as the key positions for chemerinR activation. A modified peptide (YHSFFFPGQFAFS) was synthesized and iodinated, and a radioligand binding assay was established. It was found that the ability of the various peptides to activate the chemerin receptor was strictly correlated with their affinity in the binding assay. These results confirm that a precise C-terminal processing is required for the generation of a chemerinR agonist. The possibility to restrict a medium sized protein to a nonapeptide, while keeping a low nanomolar affinity for its receptor is unusual among G protein-coupled receptors ligands. The identification of these short bioactive peptides will considerably accelerate the pharmacological analysis of chemerin-chemerinR interactions.     

巴马香猪和杜长大猪的PDK4基因的克隆及组织表达分析

为了获得广西巴马香猪和杜长大猪丙酮酸脱氢酶激酶4 (PDK4)基因编码区序列(CDS),并研究PDK4基因在广西巴马香猪和杜长大猪不同组织中的mRNA表达差异,本研究利用基因克隆技术分别从巴马香猪和杜长大猪的背最长肌组织中提取总RNA,PCR扩增出PDK4基因CDS,并进行生物信息学分析。利用实时荧光定量PCR技术检测PDK4基因在这两品种猪不同组织中m RNA的相对表达量。结果表明:本研究分别成功克隆获得巴马香猪和杜长大猪PDK4基因CDS,长度为1 224 bp,这两品种猪的PDK4基因CDS同源性为100%,与GenBank报道的普通猪、人、鼠、马、羊的同源性分别为100%、91.0%、85.5%、92.3%、93.0%。基于PDK4基因碱基同源性构建的物种系统进化树可知,巴马猪和杜长大猪遗传距离是最近的,最远的是小鼠。两品种猪PDK4氨基酸组成中亮氨酸含量较高,占总氨基酸数的10.8%,同时发现PDK4蛋白具有较强的亲水性。巴马香猪和杜长大猪PDK4蛋白的高级结构中均包含有α螺旋、无规卷曲和延伸链。实时荧光定量PCR结果显示,巴马香猪和杜长大猪的不同组织PDK4基因表达量最高的为腹脂,最低的是脾。巴马香猪背最长肌中PDK4基因表达水平极显著高于杜长大猪,而杜长大猪的皮脂、腹脂、肝、脾以及肾中PDK4基因表达水平极显著高于巴马香猪。本试验成功克隆了巴马香猪和杜长猪的PDK4基因,并且检测了该基因在两品种猪不同组织中的基因表达水平的差异,为今后深入探讨PDK4基因在地方猪种脂质代谢和脂肪沉积方面发挥的作用奠定工作基础。     

猪CFL2b基因部分序列的克隆及组织表达谱分析

在猪QTL定位基础上,利用比较基因组学原理,参照猪CFL2的cDNA序列,对猪CFL2基因的部分基因组DNA序列进行克隆、测序;利用RT-PCR半定量法检测CFL2基因在不同组织的表达情况.结果显示,所克隆的CFL2基因部分基因组DNA序列长度为2 377bp,包括4个外显子和4个内含子,该基因mRNA序列与已报道的人CFL2b基因序列相似性为89%;猪CFL2基因在多种组织中均有表达,但在骨骼肌和心肌中表达丰度较高.结果表明,本研究成功测序了猪CFL2b基因部分基因组DNA序列,为进一步研究该基因与肌肉发育及生理功能的关系奠定了基础.     

Smooth muscle myosin light chain kinase expression in cardiac and skeletal muscle

The purpose of this study was to characterize myosin light chain kinase (MLCK) expression in cardiac and skeletal muscle. The only classic MLCK detected in cardiac tissue, purified cardiac myocytes, and in a cardiac myocyte cell line (AT1) was identical to the 130-kDa smooth muscle MLCK (smMLCK). A complex pattern of MLCK expression was observed during differentiation of skeletal muscle in which the 220-kDa-long or "nonmuscle" form of MLCK is expressed in undifferentiated myoblasts. Subsequently, during myoblast differentiation, expression of the 220-kDa MLCK declines and expression of this form is replaced by the 130-kDa smMLCK and a skeletal muscle-specific isoform, skMLCK in adult skeletal muscle. These results demonstrate that the skMLCK is the only tissue-specific MLCK, being expressed in adult skeletal muscle but not in cardiac, smooth, or nonmuscle tissues. In contrast, the 130-kDa smMLCK is ubiquitous in all adult tissues, including skeletal and cardiac muscle, demonstrating that, although the 130-kDa smMLCK is expressed at highest levels in smooth muscle tissues, it is not a smooth muscle-specific protein.     

Systemic ablation of RyR3 alters Ca2+ spark signaling in adult skeletal muscle

Ca2+ sparks are localized intracellular Ca2+ release events from the sarcoplasmic reticulum in muscle cells that result from synchronized opening of ryanodine receptors (RyR). In mammalian skeletal muscle, RyR1 is the predominant isoform present in adult skeletal fibers, while some RyR3 is expressed during development. Functional studies have revealed a differential role for RyR1 and RyR3 in the overall Ca2+ signaling in skeletal muscle, but the contribution of these two isoforms to Ca2+ sparks in adult mammalian skeletal muscle has not been fully examined. When enzyme-disassociated, individual adult skeletal muscle fibers are exposed to an osmotic shock, the resting fiber converts from a quiescent to a highly active Ca2+ release state where Ca2+ sparks appear proximal to the sarcolemmal membrane. These osmotic shock-induced Ca2+ sparks occur in ryr3(-/-) muscle with a spatial distribution similar to that seen in wild type muscle. Kinetic analysis reveals that systemic ablation of RyR3 results in significant changes to the initiation, duration and amplitude of individual Ca2+ sparks in muscle fibers. These changes may reflect the adaptation of the muscle Ca2+ signaling or contractile machinery due to the loss of RyR3 expression in distal tissues, as biochemical assays identify significant changes in expression of myosin heavy chain protein in ryr3(-/-) muscle.