Skeletal muscle plays a paramount role in physical activity, metabolism, and energy balance, while its homeostasis is being challenged by multiple unfavorable factors such as injury, aging, or obesity. Exosomes, a subset of extracellular vesicles, are now recognized as essential mediators of intercellular communication, holding great clinical potential in the treatment of skeletal muscle diseases. Herein, we outline the recent research progress in exosomal isolation, characterization, and mechanism of action, and emphatically discuss current advances in exosomes derived from multiple organs and tissues, and engineered exosomes regarding the regulation of physiological and pathological development of skeletal muscle. These remarkable advances expand our understanding of myogenesis and muscle diseases. Meanwhile, the engineered exosome, as an endogenous nanocarrier combined with advanced design methodologies of biomolecules, will help to open up innovative therapeutic perspectives for the treatment of muscle diseases.
Exosomes/physiology* ; Muscle, Skeletal/metabolism* ; Cell Communication ; Homeostasis

GPR43 (G protein-coupled receptor 43) is a recently discovered short-chain free fatty acid receptor which plays important role in adipogenesis. Here we explored the transcriptional expression rule of GPR43 in porcine adipose tissue and primary cultured adipocytes. Partial cDNA of GPR43 was successfully cloned from swine by RT-PCR and the expression profile of GPR43 mRNA was studied from different types, different growing stages, and different sites of porcine adipose tissue as well as porcine primary cultured adipocytes. The results showed that porcine GPR43 shared high homology with human (89%), mouse (84%) and rat (83%). The expression level of GPR43 mRNA was significantly higher in adipose tissue of obese pigs than that of lean pigs, and also the expression level gradually increased with age. Further, the abundance of GPR43 mRNA level was higher in subcutaneous fat than in visceral fat. In addition, during the adipocytes differentiation, the expression of GPR43 mRNA increased in a time-dependent manner. These data indicated that GPR43 gene expression was relate to the site of adipose tissue, economic type, and age of pig as well as differentiating state of adipocytes, implying that GPR43 can be a potential factor to regulate adipogenesis.
Adipocytes ; cytology ; metabolism ; Adipose Tissue ; metabolism ; Animals ; Cells, Cultured ; DNA, Complementary ; biosynthesis ; genetics ; Gene Expression Profiling ; Humans ; RNA, Messenger ; biosynthesis ; genetics ; Receptors, Cell Surface ; biosynthesis ; genetics ; Receptors, G-Protein-Coupled ; biosynthesis ; genetics ; Swine ; Transcription, Genetic

In order to examine the role of astacene on mice body development and the expression of energy metabolism related genes in mice, we treated mice (Kunming white) and primary culture of mouse muscle cells with astacene of higher and lower concentration. Then the total mRNA was extracted from the muscle tissue and cells respectively, and the mRNA levels of UCP3 and LXRalpha were detected by RT-PCR in all the samples. Compared with the control group, the body weight of mice in high concentrations of astacene group grown slowly, and the expressions of UCP3 genes decreased significantly in muscle tissue of the 10th day and the 30th day as well as the cells of treated for 24 h (P<0.05). The expression of LXRalpha gene increased significantly in all samples (P<0.05) and reached its peak at 72 h (P<0.01). With the treatment of lower concentration of astacene, the expressions of UCP3 and LXRalpha gene mRNA in muscle tissue did not alter much, but in muscle cells treated for 24 h, the mRNA level of UCP3 gene decreased significantly (P<0.05), and LXRalpha gene increased significantly (P<0.05). The results suggest that astacene has a role in regulating the energy use in mice muscle.
Animals ; Carotenoids ; pharmacology ; Cells, Cultured ; Dose-Response Relationship, Drug ; Energy Metabolism ; drug effects ; genetics ; Ion Channels ; genetics ; metabolism ; Liver X Receptors ; Male ; Mice ; Mitochondrial Proteins ; genetics ; metabolism ; Muscle, Skeletal ; cytology ; metabolism ; Orphan Nuclear Receptors ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Random Allocation ; Uncoupling Protein 3

MicroRNA (miRNA) is a type of highly conserved nucleotide sequence composed of 18 to 25 nucleotides, which can specifically bind to the 3'-noncoding regions of mRNA, and then play a negative regulatory role in degrading mRNA or inhibiting translation. Long non-coding RNA (lncRNA) is a type of nucleotide sequence that exceeds 200 nucleotides in length and cannot encode proteins or can only encode protein peptides. It regulates gene expression at the levels of epigenetic, transcriptional and post-transcriptional. As an important energy storage organ, fat plays an important role in regulating the energy balance of animals, and is closely related to meat production traits such as meat production and meat quality. And the disorder of fat function can lead to hyperlipidemia, type 2 diabetes and a series of cardiovascular diseases, so the molecular regulation mechanism of animal fat deposition has attracted more attention. In recent years, more and more studies have found that miRNA and lncRNA play a crucial role in animal fat deposition. We review here the current research progresses in the role of miRNA and lncRNA in animal fat deposition, to provide theoretical guidance and new ideas for further revealing the molecular regulation mechanism of animal fat deposition.

Clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9), the third-generation genome editing tool, has been favored because of its high efficiency and clear system composition. In this technology, the introduced double-strand breaks (DSBs) are mainly repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathways. The high-fidelity HDR pathway is used for genome modification, which can introduce artificially controllable insertions, deletions, or substitutions carried by the donor templates. Although high-level knock-out can be easily achieved by NHEJ, accurate HDR-mediated knock-in remains a technical challenge. In most circumstances, although both alleles are broken by endonucleases, only one can be repaired by HDR, and the other one is usually recombined by NHEJ. For gene function studies or disease model establishment, biallelic editing to generate homozygous cell lines and homozygotes is needed to ensure consistent phenotypes. Thus, there is an urgent need for an efficient biallelic editing system. Here, we developed three pairs of integrated selection systems, where each of the two selection cassettes contained one drug-screening gene and one fluorescent marker. Flanked by homologous arms containing the mutated sequences, the selection cassettes were integrated into the target site, mediated by CRISPR/Cas9-induced HDR. Positively targeted cell clones were massively enriched by fluorescent microscopy after screening for drug resistance. We tested this novel method on the amyloid precursor protein (APP) and presenilin 1 (PSEN1) loci and demonstrated up to 82.0% biallelic editing efficiency after optimization. Our results indicate that this strategy can provide a new efficient approach for biallelic editing and lay a foundation for establishment of an easier and more efficient disease model.
Alleles ; CRISPR-Cas Systems ; DNA End-Joining Repair ; Gene Editing/methods* ; Recombinational DNA Repair

We stimulated preadipocyte of mice with calcium acetate, p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB203580, the paralysors and excitomotors of calcium channel. Then we detected expression level of preadipocyte differentiation's marker genes and calcium signal related acceptor genes by real-time PCR, and determined intracellular free Ca2+ concentration ([Ca2+]i]) with Fura-2/AM, intracellular lipid accumulation by oil red O staining. Our aim was to investigate the potential mechanism between calcium signal and preadipocyte differentiation. The results indicated that the paralysors and excitomotors of calcium channel changed the expression level of lipoprotein lipase (LPL), peroxisome proliferators-activated receptor gamma (PPARgamma), fatty acid synthetase (FAS), and the lipid accumulation, markedly. Compared with exocellular Ca2+'s decrease, inhibited intracellular Ca2+'s liberation can promoted preadipocyte differentiation (P < 0.01), and compared with intracellular Ca2+'s increase, promoted exocellular Ca2+'s ingest inhibited preadipocyte differentiation (P < 0.01). SB203580 degraded [Ca2+]i, promoted differentiation marker genes' expression and lipid accumulation in preadipocyte (P < 0.01). But calcium signal didn't have effects to vitamin D receptor (VDR) and extracellular Ca2+-sensing receptor (CaSR)'s expression. It indicated that calcium signal may effect preadipocyte different and lipid accumulation by p38 MAPK pathway.
Adipocytes ; cytology ; Animals ; Calcium ; metabolism ; Calcium Signaling ; drug effects ; Cell Differentiation ; physiology ; Cells, Cultured ; Imidazoles ; pharmacology ; Lipids ; biosynthesis ; Mice ; Pyridines ; pharmacology ; p38 Mitogen-Activated Protein Kinases ; antagonists & inhibitors

We describe a genetic transformation method of secondary somatic embryogenesis in alfalfa through cotyledon-stage somatic embryos of alfalfa infected by Agrobacterium strain GV3101. The Agrobacterium strain GV3101 contained binary vector pCAMBIA2301 that had gus gene as reporter and npt II gene as selectable marker. The infected primary embryos were induced through series of medium under 75 mg/L kanamycin selection. We obtained the transgenic alfalfa plants. Then, GUS expression in different tissue of transgenic alfalfa was tested by GUS histochemical analysis. Further, the stable integration and transformation efficiency were tested by polymerase chain reaction and Southern blotting hybridization. The result showed that GUS expression was different in different organs of transgenic alfalfa; the copy number of integrated npt II gene was from 1 to 4; the transformation efficiency via primary somatic embryogenesis was 65.82%.
Agrobacterium ; genetics ; Medicago sativa ; embryology ; genetics ; physiology ; Plant Somatic Embryogenesis Techniques ; Plants, Genetically Modified ; embryology ; genetics ; Tissue Culture Techniques ; Transformation, Genetic

OBJECTIVEIn order to investigate the possible mechanism of its function to degrade lipid, we detect the effects of hawthorn flavanone to the influence on blood-fat levels and adipogenesis genes transcription expression in fat and muscle tissue of hyperlipoidemia mouse.

METHODIn this experiment, a total of 48 mouse were randomised to four groups and irrigated with two different concentrations (1.5 g kg(-1) body weight and 3.0 g kg(-1) body weight) of hawthorn flavanone, and killed in 0 h, 1 h, 2 h and 4 h. To estimate the content of TC, TG and HCL-C in blood: Total RNA was isolated from adipose and muscle, Real-time RT-PCR was used to analyze expression changes of adipogenesis genes (SREBP-1c, FAS, HSL and TGH) with time series; to analyze the correlation between TG in blood and some kinds of adipogenesis genes and the ratio of FAS/HARMEAN (HSL, TGH) mRNA in adipose.

RESULTHawthorn flavanone was able to cut down the level ofTC, TG and HDL significantly in blood and achieved the lowest level at 1 h. In adipose tissue, hawthorn flavanone up-regulated FAS, HSL and TGH, and achieved the level of significance (P<0.05), the expression level of FAS and TGH was ascend after 1 h, but HSL descend. The expression level of SREBP-1c was descend rapidly and achieved the level of significance after treating with hawthorn flavanone at 1 h (P<0.05), after that it rise again to even higher than the level of before treatment. After treating with hawthorn flavanone, the ratio of FAS/HARMEAN (HSL, TGH) in adipose was significantly descend and achieved the lowest level at 1 h (P<0.01), but it was descendsubsequently. In muscle tissue, hawthorn flavanone was able to significantly up-regulated the expression of FAS and HSL and lower dose group showed greater increasing, the change of SREBP-1c was similar in adipose tissue except the more heavily upgrade.

CONCLUSIONHawthorn flavanone had the function of depressing the concentration of blood-fat, it co-adjusted lipid metabolism of animal by regulating the transcription expression of FAS, HSL, TGH and SREBP-1c especially HSL and SREBP-1c transcription level.

Adipose Tissue ; drug effects ; metabolism ; Animals ; Crataegus ; chemistry ; Flavanones ; pharmacology ; Gene Expression Regulation ; drug effects ; Hyperlipidemias ; blood ; genetics ; Lipids ; blood ; Lipogenesis ; drug effects ; Lipolysis ; drug effects ; genetics ; Male ; Mice ; RNA, Messenger ; genetics ; metabolism ; Sterol Regulatory Element Binding Protein 1 ; genetics ; Triglycerides ; blood ; Up-Regulation ; drug effects ; fas Receptor ; genetics

To make a universal gene targeting vector fitting for most gene and delete positive selection gene after targeting successfully, a vector named pA2T was constructed by inserting one neomycin gene (neo) for positive selection and two same herpes simplex virus thymidine kinase gene HSV-tk1 and HSV-tk2 for negative selection into the vector of pGEM-3Z, and two locus of crossing-over (x) in P1 (LoxP) and two different multiple cloning sites (MCS) were inserted into two flanks of neo separately. There were eight rare cloning sites between neo and HSV-tk1 and five rare cloning sites between neo and HSV-tk2, and neo, HSV-tk1 and HSV-tk2 could be translated respectively in the pA2T. Transfection of the pA2T into goat fetus fibroblast cells with Lipofectamine 2000 conferred resistance to geneticin (G418) and resistance to ganciclovir (GAC) in the cells, which suggested the positive and negative selectable markers could express in the cells and thus the vector pA2T could be used as a universal gene targeting vector. Transformation of the pA2T into the BM25.8 expressing Cre recombinase conferred neo was deleted in the pA2T, which suggested the LoxP was active. Thus, this vector can be inserted by most gene sequences as homologous sequences and positive selection gene can be deleted after targeting successfully, which is very convenience for the production of transgenic animals using gene targeting method.
Animals ; Animals, Genetically Modified ; genetics ; Cloning, Molecular ; Ganciclovir ; pharmacology ; Gene Targeting ; methods ; Genetic Vectors ; genetics ; Gentamicins ; pharmacology ; Goats ; Integrases ; genetics ; Neomycin ; pharmacology ; Phosphotransferases ; genetics ; metabolism