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This study aimed to elucidate the effects of maternal undernutrition (MUN) on epigenetic modification of hepatic genes in Japanese Black fetal calves during gestation. Using a previously established experimental design feeding the dams with 60% (LN) or 120% (HN) of their global nutritional requirements during the 8.5-month gestational period, DNA methylation in the fetal liver was analyzed with reduced representation bisulfite sequencing (RRBS). The promoters and gene bodies in the LN fetuses were hypomethylated compared to HN fetuses. Pathway analysis showed that the genes with DMR in the exon/intron in the LN group were associated with pathways involved in Cushing syndrome, gastric acid secretion, and aldosterone synthesis and secretion. Promoter hypomethylation in the LN group was frequently observed in genes participating in various signaling pathways (thyroid hormone, Ras/Rap1, PIK3-Akt, cAMP), fatty acid metabolism, and cholesterol metabolism. The promoter hypomethylated genes ALPL and GNAS were upregulated in the LN group, whereas the promoter hypermethylated genes GRB10 and POR were downregulated. The intron/exon hypomethylated genes IGF2, IGF2R, ACAD8, TAT, RARB, PINK1, and SOAT2 were downregulated, whereas the hypermethylated genes IGF2BP2, NOS3, and NR2F1 were upregulated. Collectively, MUN alters the promoter and gene body methylation of genes associated with hepatic metabolisms (energy, cholesterol, mitochondria) and function, suggesting an impact of altered gene methylation on the dysregulation of gene expression in the fetal liver.
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Doenças Fetais , Desnutrição , Gravidez , Humanos , Feminino , Animais , Bovinos , Metilação de DNA , Troca Materno-Fetal , Epigênese Genética , Fígado/metabolismo , Desnutrição/genética , Desnutrição/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
Skeletal muscle consists of slow and fast myofibers in which different myosin isoforms are expressed. Approximately 300 myosins form a single-thick filament in the myofibrils, where myosin is continuously exchanged. However, endogenous slow and fast myosin dynamics have not been fully understood. To elucidate those dynamics, here we generated mice expressing green fluorescence protein-tagged slow myosin heavy chain (GFP-Myh7) and Kusabira Orange fluorescence protein-tagged fast myosin heavy chain (KuO-Myh1). First, these mice enabled us to distinguish between GFP- and KuO-myofibers under fluorescence microscopy: GFP-Myh7 and KuO-Myh1 were exclusively expressed in slow myofibers and fast myofibers, respectively. Next, to monitor endogenous myosin dynamics, fluorescence recovery after photobleaching (FRAP) was conducted. The mobile fraction (Mf) of GFP-Myh7 and that of KuO-Myh1 were almost constant values independent of the regions of the myofibers and the muscle portions where the myofibers were isolated. Intriguingly, proteasome inhibitor treatment significantly decreased the Mf in GFP-Myh7 but not in KuO-Myh1 myofibers, indicating that the response to a disturbance in protein turnover depended on muscle fiber type. Taken together, the present results indicated that the mice we generated are promising tools not only for distinguishing between GFP- and KuO-myofibers but also for studying the dynamics of endogenous myosin isoforms by live-cell fluorescence imaging.
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Cadeias Pesadas de Miosina , Animais , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Miosinas/genética , Miosinas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMO
Skeletal muscles are comprised of two major types of myofibers, fast and slow. It is hypothesized that once myofiber type is determined, muscle fiber-type specificity is maintained by an epigenetic mechanism, however, this remains poorly understood. To address this, we conducted a comprehensive CpG methylation analysis with a reduced representation of bisulfite sequencing (RRBS). Using GFP-myh7 mouse, we visually distinguished and separately pooled slow-type and myh7-negative fast-type fibers for analyses. A total of 31,967 and 26,274 CpGs were hypermethylated by ≥10% difference in the fast- and slow-type fibers, respectively. Notably, the number of promoter-hypermethylated genes with downregulated expression in the slow-type fibers was 3.5 times higher than that in the fast-type fibers. Gene bodies of the fast-type-specific myofibrillar genes Actn3, Tnnt3, Tnni2, Tnnc2, and Tpm1 were hypermethylated in the slow-type fibers, whereas those of the slow-type-specific genes Myh7, Tnnt1, and Tpm3 were hypermethylated in the fast-type fibers. Each of the instances of gene hypermethylation was associated with the respective downregulated expression. In particular, a relationship between CpG methylation sites and the transcription variant distribution of Tpm1 was observed, suggesting a regulation of Tpm1 alternative promoter usage by gene body CpG methylation. An association of hypermethylation with the regulation of gene expression was also observed in the transcription factors Sim2 and Tbx1. These results suggest not only a myofiber type-specific regulation of gene expression and alternative promoter usage by gene body CpG methylation but also a dominant effect of promoter-hypermethylation on the gene expressions in slow myofibers.
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Metilação de DNA/genética , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares de Contração Lenta/metabolismo , Transcriptoma/genética , Actinina/genética , Animais , Ilhas de CpG/genética , Regulação para Baixo/genética , Epigênese Genética , Técnicas de Introdução de Genes , Ontologia Genética , Genótipo , Masculino , Camundongos , Camundongos Transgênicos , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Regiões Promotoras Genéticas , Tropomiosina/genéticaRESUMO
OBJECTIVE: On the hypothesis that grazing of cattle prompts organs to secrete or internalize circulating microRNAs (c-miRNAs) in parallel with changes in energy metabolism, we aimed to clarify biological events in adipose, skeletal muscle, and liver tissues in grazing Japanese Shorthorn (JSH) steers by a transcriptomic approach. METHODS: The subcutaneous fat (SCF), biceps femoris muscle (BFM), and liver in JSH steers after three months of grazing or housing were analyzed using microarray and quantitative polymerase chain reaction (qPCR), followed by gene ontology (GO) and functional annotation analyses. RESULTS: The results of transcriptomics indicated that SCF was highly responsive to grazing compared to BFM and liver tissues. The 'Exosome', 'Carbohydrate metabolism' and 'Lipid metabolism' were extracted as the relevant GO terms in SCF and BFM, and/or liver from the >1.5-fold-altered mRNAs in grazing steers. The qPCR analyses showed a trend of upregulated gene expression related to exosome secretion and internalization (charged multivesicular body protein 4A, vacuolar protein sorting-associated protein 4B, vesicle associated membrane protein 7, caveolin 1) in the BFM and SCF, as well as upregulation of lipolysisassociated mRNAs (carnitine palmitoyltransferase 1A, hormone-sensitive lipase, perilipin 1, adipose triglyceride lipase, fatty acid binding protein 4) and most of the microRNAs (miRNAs) in SCF. Moreover, gene expression related to fatty acid uptake and inter-organ signaling (solute carrier family 27 member 4 and angiopoietin-like 4) was upregulated in BFM, suggesting activation of SCF-BFM organ crosstalk for energy metabolism. Meanwhile, expression of plasma exosomal miR-16a, miR-19b, miR-21-5p, and miR-142-5p was reduced. According to bioinformatic analyses, the c-miRNA target genes are associated with the terms 'Endosome', 'Caveola', 'Endocytosis', 'Carbohydrate metabolism', and with pathways related to environmental information processing and the endocrine system. CONCLUSION: Exosome and fatty acid metabolism-related gene expression was altered in SCF of grazing cattle, which could be regulated by miRNA such as miR-142-5p. These changes occurred coordinately in both the SCF and BFM, suggesting involvement of exosome in the SCF-BFM organ crosstalk to modulate energy metabolism.
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OBJECTIVE: Meat quality attributes in postmortem muscle tissues depend on skeletal muscle metabolites. The objective of this study was to determine the key metabolic compounds and pathways that are associated with postmortem aging and beef quality in Japanese Black cattle (JB; a Japanese Wagyu breed with highly marbled beef). METHODS: Lean portions of Longissimus thoracis (LT: loin) muscle in 3 JB steers were collected at 0, 1, and 14 days after slaughter. The metabolomic profiles of the samples were analyzed by capillary electrophoresis time-of-flight mass spectrometry, followed by statistical and multivariate analyses with bioinformatics resources. RESULTS: Among the total 171 annotated compounds, the contents of gluconic acid, gluconolactone, spermidine, and the nutritionally vital substances (choline, thiamine, and nicotinamide) were elevated through the course of postmortem aging. The contents of glycolytic compounds increased along with the generation of lactic acid as the beef aging progressed. Moreover, the contents of several dipeptides and 16 amino acids, including glutamate and aromatic and branched-chain amino acids, were elevated over time, suggesting postmortem protein degradation in the muscle. Adenosine triphosphate degradation also progressed, resulting in the generation of inosine, xanthine, and hypoxanthine via the temporal increase in inosine 5'-monophosphate. Cysteine-glutathione disulfide, thiamine, and choline increased over time during the postmortem muscle aging. In the Kyoto encyclopedia of genes and genomes database, a bioinformatics resource, the postmortem metabolomic changes in LT muscle were characterized as pathways mainly related to protein digestion, glycolysis, citric acid cycle, pyruvate metabolism, pentose phosphate metabolism, nicotinamide metabolism, glycerophospholipid metabolism, purine metabolism, and glutathione metabolism. CONCLUSION: The compounds accumulating in aged beef were shown to be nutritionally vital substances and flavor components, as well as potential useful biomarkers of aging. The present metabolomic data during postmortem aging contribute to further understanding of the beef quality of JB and other breeds.
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Myosin is a major myofibrillar component in skeletal muscles. In myofibrils, ~300 myosin molecules form a single thick filament in which there is constant turnover of myosin. Our previous study demonstrated that the myosin replacement rate is reduced by inhibition of protein synthesis (Ojima K, Ichimura E, Yasukawa Y, Wakamatsu J, Nishimura T, Am J Physiol Cell Physiol 309: C669-C679, 2015); however, additional factors influencing myosin replacement were unknown. Here, we showed that rapid myosin replacement requires heat shock protein 90 (HSP90) activity. We utilized the fluorescence recovery after photobleaching technique to measure the replacement rate of green fluorescent protein-fused myosin heavy chain (GFP-MYH) in myotubes overexpressing HSP90. Intriguingly, the myosin replacement rate was significantly increased in HSP90-overexpressing myotubes, whereas the myosin replacement rate slowed markedly in the presence of an HSP90-specific inhibitor, indicating that HSP90 activity promotes myosin replacement. To determine the mechanism of this effect, we investigated whether HSP90 activity increased the amount of myosin available for incorporation into myofibrils. Strikingly, the gene expression levels of MYHs were significantly elevated by HSP90 overexpression but downregulated by inhibition of HSP90 activity. Cytosolic myosin content was also increased in myotubes overexpressing HSP90. Taken together, our results demonstrate that HSP90 activity facilitates myosin replacement by upregulating MYH gene expression and thereby increasing cytosolic myosin content.
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Proteínas de Choque Térmico HSP90/metabolismo , Músculo Esquelético/metabolismo , Miofibrilas/metabolismo , Miosinas/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Embrião de Galinha , Galinhas , Citosol/metabolismo , Regulação para Baixo/fisiologia , Fluorescência , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Biossíntese de Proteínas/fisiologia , Regulação para Cima/fisiologiaRESUMO
BACKGROUND: Over the past few decades, beef producers in Japan have improved marbling in their beef products. It was recently reported that marbling is not well correlated with palatability as rated by Japanese consumers. This study sought to identify the consumer segments in Japan that prefer sensory characteristics of beef other than high marbling. RESULTS: Three Wagyu beef, one Holstein beef and two lean imported beef longissimus samples were subjected to a descriptive sensory test, physicochemical analysis and a consumer (n = 307) preference test. According to consumer classification and external preference mapping, four consumer segments were identified as 'gradual high-fat likers', 'moderate-fat and distinctive taste likers', 'Wagyu likers' and 'distinctive texture likers'. Although the major trend of Japanese consumers' beef preference was 'marbling liking', 16.9% of the consumers preferred beef samples that had moderate marbling and distinctive taste. The consumers' attitudes expressed in a questionnaire survey were in good agreement with the preference for marbling among the 'moderate-fat and distinctive taste likers'. CONCLUSION: These results indicate that moderately marbled beef is a potent category in the Japanese beef market. © 2017 Society of Chemical Industry.
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Preferências Alimentares , Carne/análise , Adulto , Idoso , Animais , Bovinos , Comportamento do Consumidor , Feminino , Humanos , Japão , Masculino , Pessoa de Meia-Idade , Paladar , Adulto JovemRESUMO
Skeletal muscle tissue increases or decreases its volume by synthesizing or degrading myofibrillar proteins. The ubiquitin-proteasome system plays a pivotal role during muscle atrophy, where muscle ring finger proteins (Murf) function as E3 ubiquitin ligases responsible for identifying and targeting substrates for degradation. Our previous study demonstrated that overexpression of Ozz, an E3 specific to embryonic myosin heavy chain (Myh3), precisely reduced the Myh3 replacement rate in the thick filaments of myotubes (E. Ichimura et al., Physiol Rep. 9:e15003, 2021). These findings strongly suggest that E3 plays a critical role in regulating myosin replacement. Here, we hypothesized that the Murf isoforms, which recognize Myhs as substrates, reduced the myosin replacement rates through the enhanced Myh degradation by Murfs. First, fluorescence recovery after a photobleaching experiment was conducted to assess whether Murf isoforms affected the GFP-Myh3 replacement. In contrast to Murf2 or Murf3 overexpression, Murf1 overexpression selectively facilitated the GFP-Myh3 myosin replacement. Next, to examine the effects of Murf1 overexpression on the replacement of myosin isoforms, Cherry-Murf1 was coexpressed with GFP-Myh1, GFP-Myh4, or GFP-Myh7 in myotubes. Intriguingly, Murf1 overexpression enhanced the myosin replacement of GFP-Myh4 but did not affect those of GFP-Myh1 or GFP-Myh7. Surprisingly, overexpression of Murf1 did not enhance the ubiquitination of proteins. These results indicate that Murf1 selectively regulated myosin replacement in a Myh isoform-dependent fashion, independent of enhanced ubiquitination. This suggests that Murf1 may have a role beyond functioning as a ubiquitin ligase E3 in thick filament myosin replacement.
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Fibras Musculares Esqueléticas , Proteínas Musculares , Isoformas de Proteínas , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases , Fibras Musculares Esqueléticas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Proteínas com Motivo Tripartido/metabolismo , Proteínas com Motivo Tripartido/genética , Animais , Proteínas Musculares/metabolismo , Proteínas Musculares/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/genética , Camundongos , Cadeias Pesadas de Miosina/metabolismo , Cadeias Pesadas de Miosina/genética , Miosinas/metabolismoRESUMO
Japanese Brown (JBR) cattle have moderately marbled beef compared to the highly marbled beef of Japanese Black (JBL) cattle; however, their skeletal muscle properties remain poorly characterized. To unveil interbreed metabolic differences over the previous results, we explored the metabolome network changes before and after postmortem 7-day aging in the trapezius muscle of the two cattle breeds by employing a deep and high-coverage metabolomics approach. Using both capillary electrophoresis (CE) and ultra-high-performance liquid chromatography (UHPLC)-Fourier transform mass spectrometry (FT/MS), we detected 522 and 384 annotated peaks, respectively, across all muscle samples. The CE-based results showed that the cattle were clearly separated by breed and postmortem age in multivariate analyses. The metabolism related to glutathione, glycolysis, vitamin K, taurine, and arachidonic acid was enriched with differentially abundant metabolites in aged muscles, in addition to amino acid (AA) metabolisms. The LC-based results showed that the levels of bile-acid-related metabolites, such as tauroursodeoxycholic acid (TUDCA), were high in fresh JBR muscle and that acylcarnitines were enriched in aged JBR muscle, compared to JBL muscle. Postmortem aging resulted in an increase in fatty acids and a decrease in acylcarnitine in the muscles of both cattle breeds. In addition, metabolite set enrichment analysis revealed that JBR muscle was distinctive in metabolisms related to pyruvate, glycerolipid, cardiolipin, and mitochondrial energy production, whereas the metabolisms related to phosphatidylethanolamine, nucleotide triphosphate, and AAs were characteristic of JBL. This suggests that the interbreed differences in postmortem trapezius muscle are associated with carnitine/acylcarnitine transport, ß-oxidation, tricarboxylic acid cycle, and mitochondrial membrane stability, in addition to energy substrate and AA metabolisms. These interbreed differences may characterize beef quality traits such as the flavor intensity and oxidative stability.
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Skeletal muscle metabolism regulates homeostatic balance in animals. The metabolic impact persists even after farm animal skeletal muscle is converted to edible meat through postmortem rigor mortis and aging. Muscle metabolites resulting from animal growth and postmortem storage have a significant impact on meat quality, including flavor and color. Metabolomics studies of postmortem muscle aging have identified metabolisms that contain signatures inherent to muscle properties and the altered metabolites by physiological adaptation, with glycolysis as the pivotal metabolism in postmortem aging. Metabolomics has also played a role in mining relevant postmortem metabolisms and pathways, such as the citrate cycle and mitochondrial metabolism. This leads to a deeper understanding of the mechanisms underlying the generation of key compounds that are associated with meat quality. Genetic background, feeding strategy, and muscle type primarily determine skeletal muscle properties in live animals and affect post-mortem muscle metabolism. With comprehensive metabolite detection, metabolomics is also beneficial for exploring biomarker candidates that could be useful to monitor meat production and predict the quality traits. The present review focuses on advances in farm animal muscle metabolomics, especially postmortem muscle metabolism associated with genetic factors and muscle type.
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Farm animal skeletal muscle, formed through development, growth, and maturation processes, is converted to edible meat during postmortem rigor mortis and aging. Live and postmortem muscle metabolism is the phenotypic determinant of muscle characteristics and meat quality traits such as flavor and color. Meanwhile, animal muscle metabolism, originally programmed by genetic program, is modulated by feeding and environmental conditions through changes in the biosynthetic network. Metabolomics deepens our understanding of metabolisms underlying skeletal muscle growth, maturation, abnormality, and postmortem meat aging. The metabolomics approach is beneficial to explore biomarkers to monitor meat production processes and products, leading to improvements in livestock productivity and meat quality. One of the recent metabolomics findings in animal muscle could be the impact of mitochondrial activity and energy metabolisms on meat quality. The present review overviews the advances in metabolomics studies of farm animal skeletal muscle, to gain an insight into the muscle metabolisms associated with livestock production and meat quality.
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Animais Domésticos , Músculo Esquelético , Animais , Músculo Esquelético/metabolismo , Rigor Mortis/metabolismo , Carne/análise , MetabolômicaRESUMO
OBJECTIVE: Japanese Brown (JBR) cattle, especially the Kochi (Tosa) pedigree (JBRT), is a local breed of moderately marbled beef. Despite the increasing demand, the interbreed differences in muscle metabolites from the highly marbled Japanese Black (JBL) beef remain poorly understood. We aimed to determine flavor-related metabolites and postmortem metabolisms characteristic to JBRT beef in comparison with JBL beef. METHODS: Lean portions of the longissimus thoracis (loin) muscle from four JBRT cattle were collected at 0, 1, and 14 d postmortem. The muscle metabolomic profiles were analyzed using capillary electrophoresis time-of-flight mass spectrometry. The difference in postmortem metabolisms and aged muscle metabolites were analyzed by statistical and bioinformatic analyses between JBRT (n = 12) and JBL cattle (n = 6). RESULTS: A total of 240 metabolite annotations were obtained from the detected signals of the JBRT muscle samples. Principal component analysis separated the beef samples into three different aging point groups. According to metabolite set enrichment analysis, postmortem metabolic changes were associated with the metabolism of pyrimidine, nicotinate and nicotinamide, purine, pyruvate, thiamine, amino sugar, and fatty acid; citric acid cycle; and pentose phosphate pathway as well as various amino acids and mitochondrial fatty acid metabolism. The aged JBRT beef showed higher ultimate pH and lower lactate content than aged JBL beef, suggesting the lower glycolytic activity in postmortem JBRT muscle. JBRT beef was distinguished from JBL beef by significantly different compounds, including choline, amino acids, uridine monophosphate, inosine 5'-monophosphate, fructose 1,6-diphosphate, and betaine, suggesting interbreed differences in the accumulation of nucleotide monophosphate, glutathione metabolism, and phospholipid metabolism. CONCLUSION: Glycolysis, purine metabolism, fatty acid catabolism, and protein degradation were the most common pathways in beef during postmortem aging. The differentially expressed metabolites and the relevant metabolisms in JBRT beef may contribute to the development of a characteristic flavor.
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Calpain is an intracellular cysteine protease that cleaves its specific substrates in a limited region to modulate cellular function. Calpain-1 (C1) and calpain-2 (C2) are ubiquitously expressed in mammalian cells, but calpain-3 (C3) is a skeletal muscle-specific type. In the course of calpain activation, the N-terminal regions of all three isoforms are clipped off in an intramolecular or intermolecular fashion. C1 proteolyzes C2 to promote further proteolysis, but C2 proteolyzes C1 to suspend C1 proteolysis, indicating the presence of C1-C2 reciprocal proteolysis. However, whether C3 is involved in the calpain proteolysis network is unclear. To address this, we examined whether GFP-tagged C3:C129S (GFP-C3:CS), an inactive protease form of C3, was a substrate for C1 or C2 in HEK cells. Intriguingly, the N-terminal region of C3:CS was cleaved by C1 and C2 at the site identical to that of the C3 autoproteolysis site. Furthermore, the N-terminal clipping of C3:CS by C1 and C2 was observed in mouse skeletal muscle lysates. Meanwhile, C3 preferentially cleaved the N-terminus of C1 over that of C2, and the sizes of these cleaved proteins were identical to their autoproteolysis forms. Our findings suggest an elaborate inter-calpain network to prime and suppress proteolysis of other calpains.
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Calpaína , Músculo Esquelético , Camundongos , Animais , Calpaína/química , Calpaína/metabolismo , Proteólise , Músculo Esquelético/metabolismo , MamíferosRESUMO
This study examined the accumulation and tissue distribution of radioactive cesium nuclides in Japanese Black beef heifers raised on roughage contaminated with radioactive fallout due to the accident at the Fukushima Daiichi Nuclear Power Station on March 2011. Radiocesium feeding increased both (134)Cs and (137)Cs levels in all tissues tested. The kidney had the highest level and subcutaneous adipose had the lowest of radioactive cesium in the tissues. Different radioactive cesium levels were not found among parts of the muscles. These results indicate that radiocesium accumulated highly in the kidney and homogenously in the skeletal muscles in the heifers.
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Radioisótopos de Césio/farmacocinética , Rim/química , Músculos/química , Cinza Radioativa , Gordura Subcutânea/química , Animais , Bovinos , Radioisótopos de Césio/administração & dosagem , Feminino , Acidente Nuclear de Fukushima , Japão , Distribuição TecidualRESUMO
Myosin plays a fundamental role in muscle contraction. Approximately 300 myosins form a bipolar thick filament, in which myosin is continuously replaced by protein turnover. However, it is unclear how rapidly this process occurs and whether the myosin exchange rate differs depending on the region of the thick filament. To answer this question, we first measured myosin release and insertion rates over a short period and monitored myotubes expressing a photoconvertible fluorescence protein-tagged myosin, which enabled us to monitor myosin release and insertion simultaneously. About 20% of myosins were replaced within 10 min, while 70% of myosins were exchanged over 10 h with symmetrical and biphasic alteration of myosin release and insertion rates. Next, a fluorescence pulse-chase assay was conducted to investigate whether myosin is incorporated into specific regions in the thick filament. Newly synthesized myosin was located at the tip of the thick filament rather than the center in the first 7 min of pulse-chase labeling and was observed in the remainder of the thick filament by 30 min. These results suggest that the myosin replacement rate differs depending on the regions of the thick filament. We concluded that myosin release and insertion occur concurrently and that myosin is more frequently exchanged at the tip of the thick filament.
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Fibras Musculares Esqueléticas , Miosinas , Fibras Musculares Esqueléticas/metabolismo , Miosinas/metabolismoRESUMO
This study aimed to understand the mechanisms underlying the effects of maternal undernutrition (MUN) on liver growth and metabolism in Japanese Black fetal calves (8.5 months in utero) using an approach that integrates metabolomics and transcriptomics. Dams were fed 60% (low-nutrition; LN) or 120% (high-nutrition; HN) of their overall nutritional requirements during gestation. We found that MUN markedly decreased the body and liver weights of the fetuses; metabolomic analysis revealed that aspartate, glycerol, alanine, gluconate 6-phosphate, and ophthalmate levels were decreased, whereas UDP-glucose, UDP-glucuronate, octanoate, and 2-hydroxybutyrate levels were decreased in the LN fetal liver (p ≤ 0.05). According to metabolite set enrichment analysis, the highly different metabolites were associated with metabolisms including the arginine and proline metabolism, nucleotide and sugar metabolism, propanoate metabolism, glutamate metabolism, porphyrin metabolism, and urea cycle. Transcriptomic and qPCR analyses revealed that MUN upregulated QRFPR and downregulated genes associated with the glucose homeostasis (G6PC, PCK1, DPP4), ketogenesis (HMGCS2), glucuronidation (UGT1A1, UGT1A6, UGT2A1), lipid metabolism (ANGPTL4, APOA5, FADS2), cholesterol and steroid homeostasis (FDPS, HSD11B1, HSD17B6), and urea cycle (CPS1, ASS1, ASL, ARG2). These metabolic pathways were extracted as relevant terms in subsequent gene ontology/pathway analyses. Collectively, these results indicate that the citrate cycle was maintained at the expense of activities of the energy metabolism, glucuronidation, steroid hormone homeostasis, and urea cycle in the liver of MUN fetuses.
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Myosin, the most abundant myofibrillar protein in skeletal muscle, functions as a motor protein in muscle contraction. Myosin polymerizes into the thick filaments in the sarcomere where approximately 50% of embryonic myosin (Myh3) are replaced within 3 h (Ojima K, Ichimura E, Yasukawa Y, Wakamatsu J, Nishimura T, Am J Physiol Cell Physiol 309: C669-C679, 2015). The sarcomere structure including the thick filament is maintained by a balance between protein biosynthesis and degradation. However, the involvement of a protein degradation system in the myosin replacement process remains unclear. Here, we show that the muscle-specific ubiquitin ligase Ozz regulates replacement rate of Myh3. To examine the direct effect of Ozz on myosin replacement, eGFP-Myh3 replacement rate was measured in myotubes overexpressing Ozz by fluorescence recovery after photobleaching. Ozz overexpression significantly decreased the replacement rate of eGFP-Myh3 in the myofibrils, whereas it had no effect on other myosin isoforms. It is likely that ectopic Ozz promoted myosin degradation through increment of ubiquitinated myosin, and decreased myosin supply for replacement, thereby reducing myosin replacement rate. Intriguingly, treatment with a proteasome inhibitor MG132 also decreased myosin replacement rate, although MG132 enhanced the accumulation of ubiquitinated myosin in the cytosol where replaceable myosin is pooled, suggesting that ubiquitinated myosin is not replaced by myosin in the myofibril. Collectively, our findings showed that Myh3 replacement rate was reduced in the presence of overexpressed Ozz probably through enhanced ubiquitination and degradation of Myh3 by Ozz.
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Embrião não Mamífero/enzimologia , Proteínas Musculares/biossíntese , Miofibrilas/enzimologia , Miosinas/biossíntese , Complexos Ubiquitina-Proteína Ligase/biossíntese , Animais , Células Cultivadas , Embrião de Galinha , Citosol/enzimologia , Miosinas/antagonistas & inibidoresRESUMO
Exosomes are released from a variety of cells to communicate with recipient cells. Exosomes contain microRNAs (miRNAs), which are noncoding RNAs that suppress target genes. Our previous proteomic study (FEBS Open Bio 2016, 6, 816-826) demonstrated that 3T3-L1 adipocytes secrete exosome components as well as growth factors, inspiring us to investigate what type of miRNA is involved in adipocyte-secreted exosomes and what functions they carry out in recipient cells. Here, we profiled miRNAs in 3T3-L1 adipocyte-secreted exosomes and revealed suppression of muscle differentiation by adipocyte-derived exosomes. Through our microarray analysis, we detected over 300 exosomal miRNAs during adipocyte differentiation. Exosomal miRNAs present during adipocyte differentiation included not only pro-adipogenic miRNAs but also miRNAs associated with muscular dystrophy. Gene ontology analysis predicted that the target genes of miRNAs are associated primarily with transcriptional regulation. To further investigate whether adipocyte-secreted exosomes regulate the expression levels of genes involved in muscle differentiation, we treated cultured myoblasts with adipocyte-derived exosome fractions. Intriguingly, the expression levels of myogenic regulatory factors, Myog and Myf6, and other muscle differentiation markers, myosin heavy-chain 3 and insulin-like growth factor 2, were significantly downregulated in myoblasts treated with adipocyte-derived exosomes. Immature adipocyte-derived exosomes exhibited a stronger suppressive effect than mature adipocyte-derived exosomes. Our results suggest that adipocytes suppress the expression levels of muscle differentiation-associated genes in myoblasts via adipocyte-secreted exosomes containing miRNAs.
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Adipócitos/citologia , Exossomos/genética , Marcadores Genéticos , MicroRNAs/genética , Mioblastos/citologia , Células 3T3-L1 , Adipócitos/química , Animais , Regulação para Baixo , Perfilação da Expressão Gênica , Fator de Crescimento Insulin-Like II/genética , Camundongos , Desenvolvimento Muscular , Mioblastos/química , Fatores de Regulação Miogênica/genética , Miogenina/genética , Análise de Sequência com Séries de OligonucleotídeosRESUMO
We hypothesized that maternal low or high nutrition would give unique effects to morphological and molecular dynamics in adipose tissue of fetus of fatty breed Wagyu (Japanese Black) cattle which produce highly marbled beef. This study aimed to determine the effects of maternal energy intake in Wagyu cows, during gestation on fetal adipose tissue development, histochemical properties, and gene and microRNA (miRNA) expression. Cows were allocated to one of two nutritional energy groups: 120% (HIGH) or 60% nutritional requirements of (LOW). Fetuses (n = 6 per treatment) were removed from pregnant cows by cesarean section at fetal age 260 ± 8 days and euthanized. Subcutaneous adipose tissue (SAT), thoracic cavity visceral adipose tissue (TVAT), and perirenal adipose tissue (PAT) were collected for analysis. In histochemical analysis, in SAT and PAT, HIGH fetuses had greater diameter of adipocytes than LOW fetuses (P<0.05). Only in SAT, LOW fetuses had more Leptin (LEP) mRNA and tended to have more Peroxisome Proliferator-Activated Receptor gamma (PPARG) CCAAT-enhancer-binding proteins alpha (CEBPA) and Glucose transporter (GLUT) 4 mRNA(P<0.10). In all SAT, TVAT, and PAT, LOW fetuses had higher levels of the brown adipose tissue (BAT) biomarkers Uncoupling Protein (UCP) 1 and PPARG coactivator (PGC) 1α mRNA than HIGH fetuses (P<0.08). Meanwhile, in the other adipose tissue, LOW fetuses had lower PPARG, CEBPA, and Zinc Finger Protein (ZFP) 423 (in TVAT and PAT), FASN (in TVAT), LEP and GLUT4 mRNA (in PAT; P<0.10). In particular, in TVAT and PAT, LOW fetuses exhibited lower expression of WAT biomarkers (PPARG and ZFP423). Differential expression of various miRNAs related to adipogenesis between the LOW and HIGH fetuses was detected in an adipose tissue-specific manner (P<0.10). Based on adipose tissue-specific effects of maternal nutrition, these findings suggested that poor maternal nutrition in Wagyu cattle increased BAT development in SAT, TVAT and PAT, while elevated maternal nutrition stimulated fetal SAT development compared with that of TVAT and PAT.
Assuntos
Cesárea , MicroRNAs , Tecido Adiposo/metabolismo , Animais , Bovinos , Feminino , Feto/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
To elucidate the mechanisms underlying maternal undernutrition (MUN)-induced fetal skeletal muscle growth impairment in cattle, the longissimus thoracis muscle of Japanese Black fetal calves at 8.5 months in utero was analyzed by an integrative approach with metabolomics and transcriptomics. The pregnant cows were fed on 60% (low-nutrition, LN) or 120% (high-nutrition, HN) of their overall nutritional requirement during gestation. MUN markedly decreased the bodyweight and muscle weight of the fetus. The levels of amino acids (AAs) and arginine-related metabolites including glutamine, gamma-aminobutyric acid (GABA), and putrescine were higher in the LN group than those in the HN group. Metabolite set enrichment analysis revealed that the highly different metabolites were associated with the metabolic pathways of pyrimidine, glutathione, and AAs such as arginine and glutamate, suggesting that MUN resulted in AA accumulation rather than protein accumulation. The mRNA expression levels of energy metabolism-associated genes, such as PRKAA1, ANGPTL4, APLNR, CPT1B, NOS2, NOS3, UCP2, and glycolytic genes were lower in the LN group than in the HN group. The gene ontology/pathway analysis revealed that the downregulated genes in the LN group were associated with glucose metabolism, angiogenesis, HIF-1 signaling, PI3K-Akt signaling, pentose phosphate, and insulin signaling pathways. Thus, MUN altered the levels of AAs and expression of genes associated with energy expenditure, glucose homeostasis, and angiogenesis in the fetal muscle.