The transmembrane protein TMEM182 promotes fat deposition and alters metabolomics and lipidomics.

TMEM182, a transmembrane protein highly expressed in muscle and adipose tissues, plays a crucial role in muscle cell differentiation, metabolism, and signaling. However, its role in fat deposition and metabolism is still unknown. In this study, we used overexpression and knockout models to examine the impact of TMEM182 on fat synthesis and metabolism. Our results showed that TMEM182 overexpression increased the expression of fat synthesis-related genes and promoted the differentiation of preadipocytes into fat cells. In TMEM182 knockout mice, there was a significant decrease in abdominal fat deposition. RNA sequencing results showed that TMEM182 overexpression in preadipocytes enhanced the activity of pathways related to fat formation, ECM-receptor interaction, and cell adhesion. Furthermore, our analysis using UPLC-MS/MS showed that TMEM182 significantly altered the metabolite and lipid content and composition in chicken breast muscle. Specifically, TMEM182 increased the content of amino acids and their derivatives in chicken breast muscle, promoting amino acid metabolic pathways. Lipidomics also revealed a significant increase in the content of glycerophospholipids, sphingolipids, and phospholipids in the breast muscle after TMEM182 overexpression. These findings suggest that TMEM182 plays a crucial role in regulating fat deposition and metabolism, making it a potential target for treating obesity-related diseases and animal breeding.

Bulk and single-cell alternative splicing analyses reveal roles of TRA2B in myogenic differentiation.

Alternative splicing (AS) disruption has been linked to disorders of muscle development, as well as muscular atrophy. However, the precise changes in AS patterns that occur during myogenesis are not well understood. Here, we employed isoform long-reads RNA-seq (Iso-seq) and single-cell RNA-seq (scRNA-seq) to investigate the AS landscape during myogenesis. Our Iso-seq data identified 61,146 full-length isoforms representing 11,682 expressed genes, of which over 52% were novel. We identified 38,022 AS events, with most of these events altering coding sequences and exhibiting stage-specific splicing patterns. We identified AS dynamics in different types of muscle cells through scRNA-seq analysis, revealing genes essential for the contractile muscle system and cytoskeleton that undergo differential splicing across cell types. Gene-splicing analysis demonstrated that AS acts as a regulator, independent of changes in overall gene expression. Two isoforms of splicing factor TRA2B play distinct roles in myogenic differentiation by triggering AS of TGFBR2 to regulate canonical TGF-ß signalling cascades differently. Our study provides a valuable transcriptome resource for myogenesis and reveals the complexity of AS and its regulation during myogenesis.

Chicken muscle antibody array reveals the regulations of LDHA on myoblast differentiation through energy metabolism.

Myoblast proliferation and differentiation are highly dynamic and regulated processes in skeletal muscle development. Given that proteins serve as the executors for the majority of biological processes, exploring key regulatory factors and mechanisms at the protein level offers substantial opportunities for understanding the skeletal muscle development. In this study, a total of 607 differentially expressed proteins between proliferation and differentiation in myoblasts were screened out using our chicken muscle antibody array. Biological function analysis revealed the importance of energy production processes and compound metabolic processes in myogenesis. Our antibody array specifically identified an upregulation of LDHA during differentiation, which was associated with the energy metabolism. Subsequent investigation demonstrated that LDHA promoted the glycolysis and TCA cycle, thereby enhancing myoblasts differentiation. Mechanistically, LDHA promotes the glycolysis and TCA cycle but inhibits the ETC oxidative phosphorylation through enhancing the NADH cycle, providing the intermediate metabolites that improve the myoblasts differentiation. Additionally, increased glycolytic ATP by LDHA induces Akt phosphorylation and activate the PI3K-Akt pathway, which might also contribute to the promotion of myoblasts differentiation. Our studies not only present a powerful tool for exploring myogenic regulatory factors in chicken muscle, but also identify a novel role for LDHA in modulating myoblast differentiation through its regulation of cellular NAD+ levels and subsequent downstream effects on mitochondrial function.

Metabolomic, lipidomic, and proteomic profiles provide insights on meat quality differences between Shitou and Wuzong geese.

A comprehensive comparison of metabolomic, lipidomic, and proteomic profiles was conducted between the breast and leg muscles of Shitou goose (STE) and Wuzhong goose (WZE), which exhibit significant variations in body size and growth rate, to evaluate their impact on meat quality. WZE had higher intramuscular fat content in their breast muscles, which were also chewier and had higher drip and cooking losses than STE. Metabolomic analysis revealed differential regulation of amino acid and purine metabolism between WZE and STE. Lipidomic analysis indicated a higher abundance of PE and PC lipid molecules in WZE. Integration of proteomic and metabolomic data highlighted purine metabolism and amino acid biosynthesis as the major distinguishing pathways between STE and WZE. The primary differential pathways between breast and leg muscles were associated with energy metabolism and fatty acid metabolism. This comprehensive analysis provides valuable insights into the distinct meat quality of STE and WZE.

Natural antisense transcript of MYOG regulates development and regeneration in skeletal muscle by shielding the binding sites of MicroRNAs of MYOG mRNA 3'UTR.

Natural antisense transcripts (NATs) are endogenous RNAs opposite to sense transcripts, and they can significantly contribute to regulating various biological processes through multiple epigenetic mechanisms. NATs can affect their sense transcripts to regulate the growth and development of skeletal muscle. Our analysis of third-generation full-length transcriptome sequencing data revealed that NATs represented a significant portion of the lncRNA, accounting for up to 30.19%-33.35%. The expression of NATs correlated with myoblast differentiation, and genes expressing NATs were mainly involved in RNA synthesis, protein transport, and cell cycle. We found a NAT of MYOG (MYOG-NAT) in the data. We found that the MYOG-NAT could promote the differentiation of myoblasts in vitro. Additionally, knockdown of MYOG-NAT in vivo led to muscle fiber atrophy and muscle regeneration retardation. Molecular biology experiments demonstrated that MYOG-NAT enhances the stability of MYOG mRNA by competing with miR-128-2-5p, miR-19a-5p, and miR-19b-5p for binding to MYOG mRNA 3'UTR. These findings suggest that MYOG-NAT plays a critical role in skeletal muscle development and provides insights into the post-transcriptional regulation of NATs.

Microplastic exposure induces muscle growth but reduces meat quality and muscle physiological function in chickens.

Microplastic (MP) pollution has become one of the global environmental concerns, but the contamination and effect of MP on chicken skeletal muscle are scarcely researched. Here, we found MP contamination in the chicken skeletal muscles, which were directly collected from a large-scale chicken farm. Using Pyrolysis-Gas Chromatography-Mass Spectrometry and Agilent 8700 laser direct infrared imaging spectrometer, we found that polystyrene (PS) and polyamide are the significant type of MPs detected in chicken skeletal muscle. Constant PS-MP oral feeding for >21 days increases the content of MP deposited in chicken breast muscle, but the MP content in the leg muscle was gradually decreased. Surprisingly, the chicken's body and skeletal muscle weight was increased after constant PS-MP feeding. Physiological results showed that PS-MP exposure inhibited energy and lipid metabolism, induced oxidative stress, and potential for neurotoxicity in the skeletal muscle. Metabolomic analysis of the liquid chromatography-tandem mass spectrometry and gas chromatography coupled with the mass spectrometer results showed that PS-MP exposure changed the metabolomic profile and reduced meat quality. In vitro, experimental results showed that PS-MP exposure induced chicken primary myoblasts proliferation and apoptosis but decreased myoblasts differentiation. Transcriptome analysis of the skeletal muscle indicates that PS-MP exposure affects skeletal muscle function by regulating genes involved in neural function and muscle development. Considering that chicken is one of the most important meat foods in the world, this study will provide an essential reference for protecting meat food safety.

Transcriptome Sequencing Reveals Pathways Related to Proliferation and Differentiation of Shitou Goose Myoblasts.

Chinese Shitou goose is a type of large goose with high meat yield. Understanding the genetic regulation of muscle development in Shitou goose would be beneficial to improve the meat production traits of geese. Muscle development is regulated by genes related to myoblast proliferation and differentiation. In this study, the RNA-seq method was used to construct the mRNA and lncRNA expression profiles of Shitou goose myoblasts and myotubes. A total of 1664 differentially expressed (DE) mRNAs and 244 DE-lncRNAs were identified. The alternative mRNA splicing in proliferation and differentiation stages was also analyzed. Notably, pathways enriched in DE-mRNAs, DE-splicing transcripts, and DE-lncRNAs all point to the Wnt signaling pathway, indicating that the Wnt signaling is a key regulatory pathway of muscle development in Shitou goose. We also constructed the interactive network of DE-lncRNAs and DE-mRNAs and revealed some key genes of lncRNAs regulating the proliferation and differentiation of myoblasts. These results provide new insights for the study of the muscle development of the Shitou goose.

TMEM182 interacts with integrin beta 1 and regulates myoblast differentiation and muscle regeneration.

BACKGROUND: Transmembrane proteins are vital for intercellular signalling and play important roles in the control of cell fate. However, their physiological functions and mechanisms of action in myogenesis and muscle disorders remain largely unexplored. It has been found that transmembrane protein 182 (TMEM182) is dramatically up-regulated during myogenesis, but its detailed functions remain unclear. This study aimed to analyse the function of TMEM182 during myogenesis and muscle regeneration. METHODS: RNA sequencing, quantitative real-time polymerase chain reaction, and immunofluorescence approaches were used to analyse TMEM182 expression during myoblast differentiation. A dual-luciferase reporter assay was used to identify the promoter region of the TMEM182 gene, and a chromatin immunoprecipitation assay was used to investigate the regulation TMEM182 transcription by MyoD. We used chickens and TMEM182-knockout mice as in vivo models to examine the function of TMEM182 in muscle growth and muscle regeneration. Chickens and mouse primary myoblasts were used to extend the findings to in vitro effects on myoblast differentiation and fusion. Co-immunoprecipitation and mass spectrometry were used to identify the interaction between TMEM182 and integrin beta 1 (ITGB1). The molecular mechanism by which TMEM182 regulates myogenesis and muscle regeneration was examined by Transwell migration, cell wound healing, adhesion, glutathione-S-transferse pull down, protein purification, and RNA immunoprecipitation assays. RESULTS: TMEM182 was specifically expressed in skeletal muscle and adipose tissue and was regulated at the transcriptional level by the myogenic regulatory factor MyoD1. Functionally, TMEM182 inhibited myoblast differentiation and fusion. The in vivo studies indicated that TMEM182 induced muscle fibre atrophy and delayed muscle regeneration. TMEM182 knockout in mice led to significant increases in body weight, muscle mass, muscle fibre number, and muscle fibre diameter. Skeletal muscle regeneration was accelerated in TMEM182-knockout mice. Furthermore, we revealed that the inhibitory roles of TMEM182 in skeletal muscle depend on ITGB1, an essential membrane receptor involved in cell adhesion and muscle formation. TMEM182 directly interacted with ITGB1, and this interaction required an extracellular hybrid domain of ITGB1 (aa 387-470) and a conserved region (aa 52-62) within the large extracellular loop of TMEM182. Mechanistically, TMEM182 modulated ITGB1 activation by coordinating the association between ITGB1 and laminin and regulating the intracellular signalling of ITGB1. Myogenic deletion of TMEM182 increased the binding activity of ITGB1 to laminin and induced the activation of the FAK-ERK and FAK-Akt signalling axes during myogenesis. CONCLUSIONS: Our data reveal that TMEM182 is a novel negative regulator of myogenic differentiation and muscle regeneration.

Transcriptome profile analysis reveals KLHL30 as an essential regulator for myoblast differentiation.

Skeletal muscle development is a sophisticated multistep process orchestrated by diverse myogenic transcription factors. Recent studies have suggested that Kelch-like genes play vital roles in muscle disease and myogenesis. However, it is still unclear how Kelch-like genes impact myoblast physiology. Here, through integrative analysis of the mRNA expression profile during chicken primary myoblast and C2C12 differentiation, many differentially expressed genes were found and suggested to be enriched in myoblast differentiation and muscle development. Interestingly, a little-known Kelch-like gene KLHL30 was screened as skeletal muscle-specific gene with essential roles in myogenic differentiation. Transcriptomic data and quantitative PCR analysis indicated that the expression of KLHL30 is upregulated under myoblast differentiation state. KLHL30 overexpression upregulated the protein expression of myogenic transcription factors (MYOD, MYOG, MEF2C) and induced myoblast differentiation and myotube formation, while knockdown of KLHL30 caused the opposite effect. Furthermore, KLHL30 was found to significantly decrease the numbers of cells in the S stage and thereby depress myoblast proliferation. Collectively, this study highlights that KLHL30 as a muscle-specific regulator plays essential roles in myoblast proliferation and differentiation.

Characterization of Chicken Skin Yellowness and Exploration of Genes Involved in Skin Yellowness Deposition in Chicken.

Skin color is an important economic trait in meat-type chickens. A uniform bright skin color can increase the sales value of chicken. Chickens with bright yellow skin are more popular in China, especially in the broiler market of South China. However, the skin color of chickens can vary because of differences in breeds, diet, health, and individual genetics. To obtain greater insight into the genetic factors associated with the process of skin pigmentation in chickens, we used a colorimeter and high-resolution skin photographs to measure and analyze the skin color of chickens. By analyzing 534 chickens of the same breed, age, and feed condition, we found that the yellowness values of the chickens varied within this population. A significant positive correlation was found between the cloacal skin yellowness values before and after slaughter, and the cloacal skin yellowness value of live chickens was positively correlated with the overall body skin yellowness value. Additionally, chicken skin yellowness exhibited low heritability, ranging from 0.07 to 0.27. Through RNA sequencing, 882 genes were found to be differentially expressed between the skin with the highest and lowest yellowness values. Some of these differentially expressed genes may play an important role in yellow pigment deposition in chicken skin, which included TLR2B, IYD, SMOC1, ALDH1A3, CYP11A1, FHL2, TECRL, ACACB, TYR, PMEL, and GPR143. In addition, we found that the expression and variations of the BCO2 gene, which is referred to as the yellow skin gene, cannot be used to estimate the skin yellowness value of chickens in this population. These data will help to further our understanding of chicken skin yellowness and might contribute to the selection of specific chicken strains with consistent skin coloration.

Cytokine inducible SH2-containing protein potentiate J subgroup avian leukosis virus replication and suppress antiviral responses in DF-1 chicken fibroblast cells.

Cytokine-inducible Srchomology2 (SH2)-containing protein (CIS) belongs to the suppressors of cytokine signaling (SOCS) protein family function as a negative feedback loop inhibiting cytokine signal transduction. J subgroup avian leukosis virus (ALV-J), a commonly-seen avian virus with a feature of immunosuppression, poses an unmeasurable threat to the poultry industry across the world. However, commercial medicines or vaccines are still no available for this virus. This study aims to evaluate the potential effect of chicken CIS in antiviral response and its role on ALV-J replication. The results showed that ALV-J strain SCAU-HN06 infection induced CIS expression in DF-1 cells, which was derived from chicken embryo free of endogenous avian sarcoma-leukosis virus (ASLV) like sequences. By overexpressing CIS, the expression of chicken type I interferon (IFN-I) and interferon-stimulated genes (ISGs; PKR, ZAP, CH25H, CCL4, IFIT5, and ISG12) were both suppressed. Meanwhile, data showed that CIS overexpression also increased viral yield. Interestingly, knockdown of CIS enhanced induction of IFN-I and ISGs and inhibited viral replication. Collectively, we proved that modulation of CIS expression not only affected SCAU-HN06 replication in vitro but also altered the expression of IFN-I and ISGs that act as an essential part of antiviral innate immune system. Our data provide a potential target for developing antiviral agents for ALV-J.

Integrative Analyses of mRNA Expression Profile Reveal SOCS2 and CISH Play Important Roles in GHR Mutation-Induced Excessive Abdominal Fat Deposition in the Sex-Linked Dwarf Chicken.

Sex-linked dwarf (SLD) chicken, which is caused by a recessive mutation of the growth hormone receptor (GHR), has been widely used in the Chinese broiler industry. However, it has been found that the SLD chicken has more abdominal fat deposition than normal chicken. Excessive fat deposition not only reduced the carcass quality of the broilers but also reduced the immunity of broilers to diseases. To find out the key genes and the precise regulatory pathways that were involved in the GHR mutation-induced excessive fat deposition, we used high-fat diet (HFD) and normal diet to feed the SLD chicken and normal chicken and analyzed the differentially expressed genes (DEGs) among the four groups. Results showed that the SLD chicken had more abdominal fat deposition and larger adipocytes size than normal chicken and HFD can promote abdominal fat deposition and induce adipocyte hypertrophy. RNA sequencing results of the livers and abdominal fats from the above chickens revealed that many DEGs between the SLD and normal chickens were enriched in fat metabolic pathways, such as peroxisome proliferator-activated receptor (PPAR) signaling, extracellular matrix (ECM)-receptor pathway, and fatty acid metabolism. Importantly, by constructing and analyzing the GHR-downstream regulatory network, we found that suppressor of cytokine signaling 2 (SOCS2) and cytokine-inducible SH2-containing protein (CISH) may involve in the GHR mutation-induced abdominal fat deposition in chicken. The ectopic expression of SOCS2 and CISH in liver-related cell line leghorn strain M chicken hepatoma (LMH) cell and immortalized chicken preadipocytes (ICP) revealed that these two genes can regulate fatty acid metabolism, adipocyte differentiation, and lipid droplet accumulation. Notably, overexpression of SOCS2 and CISH can rescue the hyperactive lipid metabolism and excessive lipid droplet accumulation of primary liver cell and preadipocytes that were isolated from the SLD chicken. This study found some genes and pathways involved in abdominal fat deposition of the SLD chicken and reveals that SOCS2 and CISH are two key genes involved in the GHR mutation-induced excessive fat deposition of the SLD chicken.

Production of chiral alcohols from prochiral ketones by microalgal photo-biocatalytic asymmetric reduction reaction.

Microalgal photo-biocatalysis is a green technique for asymmetric synthesis. Asymmetric reduction of nonnatural prochiral ketones to produce chiral alcohols by microalgal photo-biocatalysis was studied in this work. Acetophenone (ACP) and ethyl acetoacetate (EAA) were chosen as model substrates for aromatic ketones and ß-ketoesters, respectively. Two prokaryotic cyanophyta and two eukaryotic chlorophyta were selected as photo-biocatalysts. The results proved that nonnatural prochiral ketones can be reduced by microalgal photo-biocatalysis with high enantioselectivity. Illumination is indispensable to the photo-biocatalysis. For aromatic ketone, cyanophyta are eligible biocatalysts. For ACP asymmetric reduction reaction, about 45% yield and 97% e.e. can be achieved by the photo-biocatalysis reaction with Spirulina platensis as biocatalyst. On the contrary, chlorophyta are efficient biocatalysts for ß-ketoester asymmetric reduction reaction among the four tested algae. For EAA asymmetric reduction reaction, about 70% yield and 90% e.e. can be achieved with Scenedesmus obliquus as biocatalyst. The microalgae used in this study outperformed other characterized biocatalysts such as microbial and plant cells.