RESUMO
OBJECTIVE: Considerable uncertainty remains regarding the veracity of measuring myokine irisin more than seven years after its original description. Unresolved issues include the nature of transcription of the irisin precursor fibronectin type III domain containing 5 (FNDC5) gene across species, the reliability of irisin levels measured with commercial enzyme-linked immunosorbent assays (ELISAs), and the overall validity of the recently published reference values for human serum measured with quantitative mass spectrometry. We utilized multiple species and measures to evaluate the robustness of commonly used reagents and methods for reporting irisin. METHODS: Amplification of cDNA was used to assess the FNDC5 transcript patterns in humans and mice. The specificity and sensitivity of different irisin antibodies were examined via western blotting. Quantification of circulating native irisin was conducted with mass spectrometry using an absolute quantification peptide for irisin. RESULTS: We show that there is a greater transcript diversity of human FNDC5 than currently annotated, but no indication of the expression of transcripts leading to a truncated form of irisin. Available irisin antibodies still bind to patterns of unspecific serum proteins, which compromise reliable measurements of irisin with ELISAs. Absolute quantification of irisin with labeled peptides by mass spectrometry is an advanced method but requires a multi-step sample preparation introducing uncontrollable variations in the measurement. CONCLUSION: Our data represent an explicit warning against measuring circulating irisin using available methods. Measuring irisin is akin to chasing shadows.
Assuntos
Fibronectinas/metabolismo , Músculos/metabolismo , Animais , Equidae , Fibronectinas/sangue , Fibronectinas/genética , Cabras , Humanos , Espectrometria de Massas , Camundongos , Papio , Coelhos , RatosRESUMO
Unlike specific expression in the skin of wild mice, the agouti signaling protein (ASIP) is expressed widely in the tissue of cattle, including adipose and muscle tissue. Hence, it has been suggested that ASIP plays a role in bovine fat metabolism. An inserted L1-BT element was recently identified upstream of the ASIP locus which led to an ectopic expression of ASIP mRNA in cattle. In this study, we detected the indel of the L1-BT element at g. - 14 643 â¯nt and three SNPs in introns of the ASIP gene (g. - 568 A⯠> â¯G, g. - 554 A⯠> â¯T, and g. 4805A⯠> â¯T) in a Chinese Simmental steer population. The association analysis between variants of ASIP and economic traits showed that the homozygous genotype of L1-BT element insertion, AA genotype of g. - 568 A⯠> â¯G, and AT genotype of g. 4805A⯠> â¯T were significantly correlated with carcass and fat-related traits, such as live weight and back fat thickness. Moreover, three haplotypes (H1: AT; H2: AA; H3: GT) were identified by linkage disequilibrium analysis and formed six combined genotypes. Results indicated that Chinese Simmental steers with an H1H2 combined genotype had a higher measured value of fat-deposition-related traits ( p < 0.05 ), including thickness of back fat and percentage of carcass fat coverage, but a lower content of linoleic acid and α -linolenic acid ( p < 0.05 ). Individuals of an H3H3 combination had a lower marbling score, perirenal fat weight, and carcass weight ( p < 0.05 ). This suggests that these three SNPs and two combined haplotypes might be molecular markers for beef cattle breeding selection.
RESUMO
Transcriptome analyses of bovine muscle tissue differing in intramuscular fat (IMF) content identified agouti signaling protein (ASIP) as a promising candidate gene for fat deposition. The protein is secreted from adipocytes and may serve as a signaling molecule in cross-talk between adipocytes and muscle fibers or other cells. Known receptors for ASIP are the melanocortin receptors (e.g., MC4R) and attractin (ATRN). The present study was conducted to determine relationships between the expression of ASIP and its receptors in different bovine tissues with fat deposition. Adipose tissues, liver, and longissimus muscle tissue were collected from 246 F2-generation bulls (Charolais × Holstein cross) and gene expression was measured with RT-qPCR. During analysis of subcutaneous fat (SCF) of all bulls, 17 animals were identified with a transposon-derived transcript (Exon2C) inserted in the ASIP gene and dramatically increased ASIP mRNA levels. Significant correlations between normalized mRNA values of SCF and phenotypic traits related to fat deposition were found in bulls without Exon2C. Three retrospectively assigned groups [Exon2C, n = 17; high carcass fat (HCF), n = 20; low carcass fat (LCF), n = 20] were further analyzed to verify expression differences and elucidate molecular reasons. Expression of ASIP could be detected in isolated muscle fibers and adipocytes of Exon2C bulls in contrast to HCF and LCF bulls, indicating ectopic ASIP expression if the transposon is present. Among adipose tissues, highest ASIP mRNA levels were measured in SCF with significantly higher values in HCF compared to LCF bulls (1.6-fold, P < 0.05). However, the protein abundance was below the detection limit in all bulls. Potential ASIP receptors were detected in most investigated tissues. The expression of MC4R was higher and of ATRN was lower in several tissues of LCF compared to HCF bulls, whereas MC1R was not differentially expressed. Bulls of the Exon2C group had lower ATRN mRNA values than HCF and LCF bulls in perirenal fat (PF), but higher (P < 0.05) values in muscle. Receptors were also expressed in tissues where ASIP mRNA was not detected. Consequently, those tissues could be targets for ASIP if it circulates.
RESUMO
Thyroid hormone responsive protein (THRSP) is known to be involved in lipogenic processes in rodents. In cattle, THRSP could be a potential molecular marker for intramuscular fat (IMF) deposition since mRNA abundance was frequently found to be increased in skeletal muscle with high IMF content compared to those with low IMF. The aim of this study was to elucidate the background of this differential expression and to evaluate the role of THRSP as candidate for increased IMF content in cattle. By combination of mRNA and protein analyses, we could demonstrate that THRSP is present mainly in nuclei of adipose tissue, in intramuscular fat cells and associated cells, and in cells of the portal triad of liver, whereas muscle cells did not express THRSP. Cell culture analyses revealed furthermore that THRSP is expressed in mature adipocytes rather than in early stages of adipogenesis. Collectively, our data support the putative role of THRSP as transcriptional regulator and demonstrate that an increased expression of THRSP in M. longissimus is a consequence of but not the reason for a higher number of intramuscular adipocytes in cattle with enhanced IMF deposition.
Assuntos
Músculo Esquelético/metabolismo , Fatores de Transcrição/metabolismo , Células 3T3-L1 , Animais , Bovinos , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/fisiologia , Metabolismo dos Lipídeos/genética , Metabolismo dos Lipídeos/fisiologia , Camundongos , RNA Mensageiro/metabolismo , Fatores de Transcrição/genéticaRESUMO
Adipose tissue and skeletal muscle are organs that respond strongly to obesity and physical activity exhibiting high secretory activity. To identify novel putative adipomyokines, comparative expression studies of skeletal muscle and adipose tissue of lean (C57BL/6J) and obese (C57BL/6J on a high-fat diet and NZO) mice, of sedentary and endurance trained C57BL/6J mice and of cattle characterized by different amounts of intramuscular fat were combined with human secretome data and scored. In highly regulated transcripts, we identified 119 myokines, 79 adipokines and 22 adipomyokines. Network analysis of these candidates revealed remodelling of extracellular matrix and tissue fibrosis as relevant functions of several of these candidates. Given the pathophysiogical relevance of fibrosis for adipose-muscle-cross-talk in obesity and type 2 diabetes and its physiological role in exercise adaptation and meat quality of farm animals, they represent interesting candidates for further investigations in different research areas and species.
Assuntos
Adipocinas/metabolismo , Tecido Adiposo/metabolismo , Citocinas/metabolismo , Músculo Esquelético/metabolismo , Obesidade/metabolismo , Proteoma , Transcriptoma , Adipocinas/genética , Tecido Adiposo/citologia , Animais , Bovinos , Células Cultivadas , Citocinas/genética , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Redes Reguladoras de Genes , Humanos , Canais Iônicos/fisiologia , Masculino , Síndrome Metabólica/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Mitocondriais/fisiologia , Músculo Esquelético/citologia , Obesidade/etiologia , Condicionamento Físico Animal , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade da Espécie , Proteína Desacopladora 1RESUMO
The aim of this study was to develop adequate in vitro conditions for the differentiation of bovine skeletal muscle cells. Therefore, satellite cells isolated from the left foreleg of a Holstein-Friesian fetus at 4.5 mo of gestation were seeded on 24-well plates coated with extracellular matrix gel. Cells were cultured for 5 d in growth medium containing 10% fetal bovine serum. After reaching confluence, several differentiation media were tested for inducing myotube formation. The highest fusion rate of approximately 30% was achieved with a serum-free medium containing 1 µM dexamethasone, 1 µg/ml linoleic acid, and 0.1 µM insulin after a differentiation phase of 72 h. Two different culture conditions (serum-free and serum-containing) appropriate for bovine skeletal muscle cell differentiation are described in detail which allow the investigation of bovine skeletal muscle cell proliferation and differentiation in general as well as in response to bioactive compounds.
Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular , Músculo Esquelético/citologia , Mioblastos/citologia , Células Satélites de Músculo Esquelético/citologia , Animais , Bovinos , Músculo Esquelético/embriologiaRESUMO
The myokine irisin is supposed to be cleaved from a transmembrane precursor, FNDC5 (fibronectin type III domain containing 5), and to mediate beneficial effects of exercise on human metabolism. However, evidence for irisin circulating in blood is largely based on commercial ELISA kits which are based on polyclonal antibodies (pAbs) not previously tested for cross-reacting serum proteins. We have analyzed four commercial pAbs by Western blotting, which revealed prominent cross-reactivity with non-specific proteins in human and animal sera. Using recombinant glycosylated and non-glycosylated irisin as positive controls, we found no immune-reactive bands of the expected size in any biological samples. A FNDC5 signature was identified at ~20â kDa by mass spectrometry in human serum but was not detected by the commercial pAbs tested. Our results call into question all previous data obtained with commercial ELISA kits for irisin, and provide evidence against a physiological role for irisin in humans and other species.
Assuntos
Artefatos , Ensaio de Imunoadsorção Enzimática/métodos , Exercício Físico/fisiologia , Fibronectinas/sangue , Músculo Esquelético/metabolismo , Animais , Análise Química do Sangue/métodos , Citocinas/sangue , Humanos , Camundongos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Recent findings regarding the response of fibronectin type III domain-containing protein 5 (Fndc5) and irisin to exercise are partly controversial. While the 25 kDa form of Fndc5 can be observed in muscle and serum of different species, the ~12 kDa irisin band was not detectable up to now. The present study aimed to clarify whether irisin exists in its theoretical size of ~12 kDa in mice and if it is affected by exercise. Male mice were randomly assigned to a sedentary control group (CO), a group with free access to running wheels (RW), and a treadmill group (TM). Blood and leg muscles were collected to investigate the regulatory cascade including peroxisome proliferator-activated receptor gamma co-activator 1-alpha (Ppargc1a) and Fndc5. In western blot analysis, antibodies were used capable of differentiation between full-length Fndc5 and irisin. This enabled us to demonstrate that irisin exists in muscle and serum of mice independent of exercise and that it is increased immediately after acute exercise. Different transcripts of Ppargc1a mRNA, but not Fndc5 mRNA, were up-regulated in the TM group. Furthermore, neither Fndc5 (25 kDa) nor Ppargc1a protein was elevated in muscle tissue. The Ppargc1a-Fndc5/irisin pathway did not clearly respond to mild exercise in the RW group. Our results provide evidence for the existence of irisin and for its immediate response to acute exercise in mice.
Assuntos
Fibronectinas/sangue , Músculo Esquelético/metabolismo , Condicionamento Físico Animal/fisiologia , Animais , Imuno-Histoquímica , Masculino , Camundongos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Distribuição Aleatória , Fatores de Transcrição/metabolismoRESUMO
The transmembrane protein FNDC5 was recently characterized as precursor of an exercise induced myokine named irisin. Previous studies found a relationship between circulating irisin levels and muscle mass in humans. Consequently, we tested the hypothesis whether FNDC5/irisin is involved in the modulation of body composition in cattle. Since information on the bovine FNDC5 locus was scarce, we characterized the gene experimentally as prerequisite for these investigations. We provide here a revised and extended gene model for bovine FNDC5. Although similarly organized like the human and murine loci, a higher variability was observed at transcript level in the bovine locus. FNDC5 mRNA was abundant in bovine skeletal muscle and was detected at lower levels in adipose tissue and liver. There were no expression differences between two groups of bulls highly different in muscularity and adiposity. Full-length FNDC5 protein (25 kDa) was present in bovine skeletal muscle independent of muscularity. Neither FNDC5 nor its putatively secreted peptide irisin were found in circulation of bulls. In contrast, we demonstrated that FNDC5 (25 kDa) and irisin (12 kDa) were present in murine skeletal muscle and that irisin was circulating in murine serum. This indicates fundamental differences in the regulation of FNDC5 and irisin between rodents and cattle.