Regulation of cardiac and skeletal muscle protein synthesis by individual branched-chain amino acids in neonatal pigs

Skeletal muscle grows at a very rapid rate in the neonatal pig, due in part to an enhanced sensitivity of protein synthesis to the postprandial rise in amino acids. An increase in leucine alone stimulates protein synthesis in skeletal muscle of the neonatal pig; however, the effect of isoleucine and valine has not been investigated in this experimental model. The left ventricular wall of the heart grows faster than the right ventricular wall during the first 10 days of postnatal life in the pig. Therefore, the effects of individual BCAA on protein synthesis in individual skeletal muscles and in the left and right ventricular walls were examined. Fasted pigs were infused with 0 or 400 micromol x kg(-1) x h(-1) leucine, isoleucine, or valine to raise individual BCAA to fed levels. Fractional rates of protein synthesis and indexes of translation initiation were measured after 60 min. Infusion of leucine increased (P < 0.05) phosphorylation of eukaryotic initiation factor (eIF)4E-binding protein-1 and increased (P < 0.05) the amount and phosphorylation of eIF4G associated with eIF4E in longissimus dorsi and masseter muscles and in both ventricular walls. Leucine increased (P < 0.05) the phosphorylation of ribosomal protein (rp)S6 kinase and rpS6 in longissimus dorsi and masseter but not in either ventricular wall. Leucine stimulated (P < 0.05) protein synthesis in longissimus dorsi, masseter, and the left ventricular wall. Isoleucine and valine did not increase translation initiation factor activation or protein synthesis rates in skeletal or cardiac muscles. The results suggest that the postprandial rise in leucine, but not isoleucine or valine, acts as a nutrient signal to stimulate protein synthesis in cardiac and skeletal muscles of neonates by increasing eIF4E availability for eIF4F complex assembly.     

Molecular characterization and expression analysis of caveolin-1 in pig tissues

Members of the caveolin family played important roles during fundamental cellular processes,such as regulation of cell morphology,migration,and gene expression in muscle cells.In this study,caveolin-1 (Cav-1),one of the caveolins,was identified from longissimus dorsi muscle of Large Yorkshire pig and Chinese indigenous Lantang pig based on the results of mRNA differential display analysis.The deduced amino acids sequence of the porcine Cav-1 contained a caveolin domain,and was very conservative among different species.The Cav-1 mRNA was widely expressed in the eight tissues in this study,including heart,liver,kidney,encephalon,spleen,lung,longissimus dorsi muscle,and back fat, and the highest expression quantity was found in back fat of the two pig breeds.The expression quantity of porcine Cav-1 in back fat and longissimus dorsi muscle of Lantang pig was significantly higher than that of Large Yorkshire(P<0.01,and P<0.05,respectively).These results suggested that the Cav-1 might be a candidate gene for carcass traits,and might provide valuable information for understanding the mechanism of caveolae signaling in fat deposition by using the animal model of pig.     

猪骨骼肌快肌肌钙蛋白C2基因的cDNA克隆与表达分析

从人骨骼肌快肌肌钙蛋白C2（TNNC2）基因出发,在dbEST数据库中进行同源性搜索,找到一个有较高同源性且在猪背最长肌中表达EST（BM083186）。通过电子克隆和进一步RT-PCR实验验证,获得猪TNNC2基因全长cDNA序列,其全长843bp,开放阅读框为201～683bp,编码有160个氨基酸。同源性分析结果表明,与人、鼠的骨骼肌快肌肌钙蛋白C2基因cDNA编码区（CDS）同源性分别为93.6％、90.5％,蛋白序列同源性均为97.5%。多种组织的半定量RT-PCR研究表明,该基因在骨骼肌中表达,并且在杜洛克猪背最长肌中的表达比兰塘猪高。     

Relationships between mitochondrial content and glycogen distribution in porcine muscle fibres.

Serial sections of longissimus dorsi and rectus femoris muscles from 15 Yorkshire breed pigs (live weights 24-46 and 49-139 kg) were stained for glycogen (PAS) and a mitochondrial enzyme (NAD tetrazolium reductase). Muscle fibres with a low mitochondrial content in both muscles were more frequently PAS-positive than fibres with a high or intermediate mitochondrial content. However, some pigs had all their muscle fibres PAS-positive while one pig with a high post-mortem muscle pH had all rectus femoris fibres PAS-negative. Relative to lighter weight pigs, longissimus dorsi muscles of heavy pigs tended to have less fibres with a high mitochondrial content and less fibres with a positive PAS reaction. Compared to longissimus dorsi muscles, rectus femoris muscles had more fibres with a high mitochondrial content and less with a positive PAS reaction. All fibres in both muscles became PAS-negative with an accompanying decrease in pH by 24 hr post-mortem. Fibres from longissimus dorsi muscles frequently had PAS-positive sarcoplasmic cores between their myofibrils. Heavy pigs tended to have larger cores (up to a mean maximum diameter of 13.4 mum), more fibres with cores, and more cores per fibre. The pigs involved exhibited no other ante- or post-mortem muscle abnormalities.     

Association of 3 polymorphisms in porcine troponin I genes (TNNI1 andTNNI2) with meat quality traits

The contractile protein troponin I (TnI), a constituent protein of the troponin complex located on the thin filaments of striated muscle, is involved in inhibition of calcium-induced myosin AT Pase activity (and thus contraction). TnI-slow (slow-twitch skeletal muscle isoform, named TNNI1) and TnI-fast (fast-twitch skeletal muscle isoform, named TNNI2) are muscle-fiber-type-specific proteins, and expression of their genes may affect the composition of muscle fiber, thereby influencing the meat quality traits. Thus, the TnI genes are potential candidate genes for traits related to meat quality in animals. Association of 2 SNPs (EU743939:g.5174T>C in intron 4, and EU743939:g.8350C>A in intron 7) of theTNNI1 gene and a SNP (EU696779:g.1167C>T in intron 3) of theTNNI2 gene with 11 meat quality traits were studied on 334 Large White × Meishan F₂ pigs. In theTNNI1 gene, g.5174T>C and g.8350C>A were found to be significantly associated with intramuscular fat content and meat color value of biceps femoris. The g.5174T>C also showed significant effects on meat color value and marbling score of longissimus dorsi, as well as pH of longissimus dorsi and semispinalis capitis. The g.1167C>T polymorphism in theTNNI2 gene affected significantly the pH of longissimus dorsi, meat color value of longissimus dorsi and semispinalis capitis, marbling score of longissimus dorsi, and intramuscular fat.     

Expression and genome polymorphism of ACSL1 gene in different pig breeds

Acyl coenzyme A long-chain 1 synthetase (ACSL1) plays a key role in animal fat synthesis and fatty acid β-oxidation. In order to research the function of the ACSL1 gene in pig, we analyzed the mRNA expression in liver, backfat and longissimus dorsi muscle by quantitative real-time PCR in Tibet pig (TP, n = 10), Diannan small ear pig (DSP, n = 10) and large white pig (LW, n = 10). The results showed that the mRNA expressions of the ACSL1 gene in liver and longissimus dorsi muscle of DSP and TP were significant higher than that of LW (P < 0.01). However, the expression in backfat of LW was significant higher than that of TP (P < 0.01) and DSP (P < 0.05). In addition, four SNPs located in 5' flanking region (T-1191C), exon 6(G173A), exon 14(C36T) and exon 17(T46C) were identified, and the allele frequencies of the four SNPs were significant different in indigenous and introduced pig breeds. The results indicated that the ACSL1 gene might be relative to the capacity of fat deposition and meat quality in pig breeds.     

Electrophoretic differentiation of myofibrillar proteins in the pig

1. Starch-gel electrophoretograms of myosin and tropomyosin preparations in 8m-urea, from longissimus dorsi and psoas muscles of the pig, were characterized by laser densitometry. 2. The typical pattern for freshly prepared myosin from both muscles was similar, there being five electrophoretically distinct components. 3. The number of electrophoretically distinct components in both muscles increased after freeze-drying, but the effect of freeze-drying was more marked in longissimus dorsi. 4. Extraction with 8m-urea containing 2% β-mercaptoethanol decreased the number of major electrophoretically distinct components of the fresh myosin of both muscles to four. 5. Although there was also some simplification of the patterns after freeze-drying the greater susceptibility of the myosin from longissimus dorsi was still evident. 6. The typical pattern for freshly prepared tropomyosin in 8m-urea differed in the two muscles: in each case it was more complex than that of the corresponding myosins. 7. The pattern of tropomyosin from neither longissimus dorsi nor psoas was altered significantly after freeze-drying. 8. Electrophoretograms of pig longissimus dorsi tropomyosin in 8m-urea differed from those of longissimus dorsi tropomyosin from sheep, ox and rabbit. 9. Extraction of the tropomyosins in 8m-urea and 2% β-mercaptoethanol simplified the electrophoretic pattern to two major components with samples from pig, sheep and ox, and to one major component with samples from rabbit. 10. It was concluded that classification of skeletal muscles as `red' or `white' is insufficient to account for the degree of functional specialization which the electrophoretograms suggest.     

Functional study of Villin 2 protein expressed in longissimus dorsi muscle of Korean native cattle in different growth stages

The aim of this study was to investigate protein profiles related to the induction of adipogenesis within the bovine longissimus dorsi muscle (BLDM) by proteomic analysis. We analyzed BLDM proteins at different growth stages to clarify the physiological mechanisms of marbled muscle development in 20 head of Korean native cattle (11 month: 10 head, 17 month: 10 head). BLDM proteins were analyzed by two-dimensional electrophoresis and image analysis. Villin 2 was specifically identified by mass spectrometry and a protein search engine. Villin 2 protein expression in BLDM decreased during the fat development stage in test steers. In a Western blot cell culture study of spontaneously immortal bovine muscle fibroblasts, the abundance of Villin 2 was shown to be down-regulated during differentiation into muscle. In 3T3-L1 mouse embryonic fibroblasts, Villin 2 was decreased during differentiation into adipocytes. The results suggest that Villin 2 may be related to the induction of transdifferentiation and adipogenesis in bovine longissimus dorsi muscle.     

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。     

Molecular cloning,sequence analysis and tissue-specific expression of Akirin2 gene in Tianfu goat

The Akirin2 gene is a nuclear factor and is considered as a potential functional candidate gene for meat quality. To better understand the structures and functions of Akirin2 gene, the cDNA of the Tianfu goat Akirin2 gene was cloned. Sequence analysis showed that the Tianfu goat Akirin2 cDNA full coding sequence (CDS) contains 579 bp nucleotides that encode 192 amino acids. A phylogenic tree of the Akirin2 protein sequence from the Tianfu goat and other species revealed that the Tianfu goat Akirin2 was closely related with cattle and sheep Akirin2. RT-qPCR analysis showed that Akirin2 was expressed in the myocardium, liver, spleen, lung, kidney, leg muscle, abdominal muscle and the longissimus dorsi muscle. Especially, high expression levels of Akirin2 were detected in the spleen, lung, and kidney whereas lower expression levels were seen in the liver, myocardium, leg muscle, abdominal muscle and longissimus dorsi muscle. Temporal mRNA expression showed that Akirin2 expression levels in the longissimus dorsi muscle, first increased then decreased from day 1 to month 12. Western blotting results showed that the Akirin2 protein was only detected in the lung and three skeletal muscle tissues.     

Changes in the expression of uncoupling proteins and lipases in porcine adipose tissue and skeletal muscle during feed deprivation*(1)

The hormone-sensitive and lipoprotein lipases are critical determinants of the metabolic adaptation to starvation. Additionally, the uncoupling proteins have emerged with potential roles in the metabolic adaptations required by energy deficiency. The objective of this study was to evaluate the expression (mRNA abundance) of uncoupling proteins 2 and 3 and that of hormone-sensitive and lipoprotein lipase in the adipose tissue and skeletal muscle of the pig in relationship to feed deprivation. Thirty-two male castrates (87 kg +/- 5%) were assigned at random to fed and feed-deprived treatment groups. After 96 hr, the pigs were euthanized and adipose and skeletal muscle tissue obtained for total RNA extraction and nuclease protection assays. Feed deprivation increased uncoupling protein 3 mRNA abundance 103-237% (P < 0.01) in longissimus and red and white semitendinosus muscle. In contrast, the increase in uncoupling protein 3 mRNA in adipose tissue was only 23% (P < 0.06), and adipose uncoupling protein 2 mRNA was not influenced (P > 0.66) by feed deprivation. The increased abundance of uncoupling protein 2 mRNA in the longissimus muscle of feed-deprived pigs was small (22%), but significant (P < 0.04). The expression of hormone-sensitive lipase was increased 46% and 64% (P < 0.04) in adipose tissue and longissimus muscle, respectively, by feed deprivation, whereas adipose lipoprotein lipase expression was reduced (P < 0.01) to 20% of that of the fed group. Longissimus lipoprotein lipase expression in the feed-deprived group was 37% of that of the fed group (P < 0.01), and similar reductions were detected in red and white semitendinosus muscle. Overall, these findings indicate that uncoupling protein 3 expression in skeletal muscle is quite sensitive to starvation in the pig, whereas uncoupling protein 2 changes are minimal. Furthermore, we conclude that hormone-sensitive lipase is upregulated at the mRNA level with prolonged feed deprivation, whereas lipoprotein lipase is downregulated.     

Molecular cloning, sequence identification and expression analysis of novel caprine MYLPF gene

Skeletal muscle genes are important potentially functional candidate genes for livestock production and meat quality. Myosin regulatory light chain (MLC) regulates myofilament activation via phosphorylation by Ca²⁺ dependent myosin light chain kinase. The cDNA of the myosin light chain, phosphorylatable, fast skeletal muscle (MYLPF) gene from the longissimus dorsi of Tianfu goat was cloned and sequenced. The results showed that MYLPF full-length coding sequence consists of 513 bp and encodes 170 amino acids with a molecular mass of 19.0 kD. Two EF-hand superfamily domain of MYLPF gene conserved between caprine and other animals. The deduced amino acid sequence of MYLPF shared significant identity with the MYLPF from other mammals. A phylogenetic tree analysis revealed that the caprine MYLPF protein has a close genetic relationship and evolutional distance with MYLPF in other mammals. Analysis by RT-PCR showed that the MYLPF mRNA was detected in heart, liver, spleen, lung, kidney, gastrocnemius, abdominal muscle and longissimus dorsi. In particular, high expression levels of MYLPF mRNA were detected in the longissimus dorsi, gastrocnemius and abdominal muscle, and low level of expressions were observed in liver, spleen, lung and kidney. In addition, the temporal expression analysis further showed MYLPF expression decreased gradually with age in the skeletal muscle. This may be important as muscle growth occurs mainly in young age in goats. Western blotting results detected the MYLPF protein in four of the tissues in which MYLPF was shown to be expressed; the four exceptions were liver, spleen, lung and kidney.     

Quantitative trait loci for fatty acid composition in longissimus dorsi and abdominal fat: results from a White Duroc × Erhualian intercross F2 population

A whole-genome scan was performed on 660 F₂ animals including 250 barrows and 410 gilts in a White Duroc × Erhualian intercross population to detect quantitative trait loci (QTL) for fatty acid composition in the longissimus dorsi muscle and abdominal fat. A total of 153 QTL including 63 genome-wide significant QTL and 90 suggestive effects were identified for the traits measured. Significant effects were mainly evident on pig chromosomes (SSC) 4, 7, 8 and X. No association was detected on SSC3 and 11. In general, the QTL detected in this study showed distinct effects on fatty acid composition in the longissimus muscle and abdominal fat. The QTL for fatty acid composition in abdominal fat did not correspond to those identified previously in backfat and the majority of QTL for the muscle fatty acid composition were mapped to chromosomal regions different from previous studies. Two regions on SSC4 and SSC7 showed significant pleiotropic effects on monounsaturated (MUFA) and polyunsaturated fatty acid (PUFA) in both longissimus muscle and abdominal fat. Another two QTL with significant multi-faceted effects on MUFA and PUFA in the longissimus muscle were found each on SSC8 and SSCX. Chinese Erhualian alleles were associated with increased ratios of MUFA to saturated fatty acid at most of the QTL detected, showing beneficial effect in terms of human health.     

Molecular characterization and expression profile of a novel porcine gene differentially expressed in the muscle and backfat tissues from Chinese Meishan and Russian Large White pigs

The mRNA differential display technique was performed to investigate the differences of gene expression in the longissimus dorsi muscle and backfat tissues from Chinese Meishan and Russian Large White pigs. One novel gene that was differentially expressed was identified through semi-quantitative RT-PCR and the cDNA complete sequence was then obtained using the rapid amplification of cDNA ends (RACE) method. The cDNA sequence of this gene is not homologous to any of the known porcine genes. The sequence prediction analysis revealed that the open reading frame of this gene encodes a protein of 402 amino acids that contains the putative conserved transposase DDE domain and further Blast analysis revealed that this protein has 100% homology with the Tn10 transposase from Oryza sativa, Serratia marcescens, and Salmonella, and therefore, this gene can be defined as the swine Tn10 transposase gene. This novel porcine gene was finally assigned to Gene ID: 100049649. The RT-PCR analysis of the tissue expression profile was carried out using the tissue cDNAs of one Meishan pig as the templates, and the result indicated that this novel swine gene is moderately expressed in fat, and weakly expressed in small intestine, liver, kidney, and spleen but almost not expressed in heart, ovary, muscle, and lung. Our experiment established the primary foundation for further research into the biological significance of swine Tn10 transposase gene.