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

The growth and development of skeletal muscle is an important factor affecting pork production and quality, which is elaborately regulated by many genetic and nutritional factors. MicroRNA (miRNA) is a non-coding RNA with a length of about 22 nt, which binds to the 3'UTR sequence of the mRNA of the target genes, and consequently regulates its post-transcriptional expression level. In recent years, a large number of studies have shown that miRNAs are involved in various life processes such as growth and development, reproduction, and diseases. The role of miRNAs in the regulation of porcine skeletal muscle development was reviewed, with the hope to provide a reference for the genetic improvement of pigs.
Swine ; Animals ; MicroRNAs/metabolism* ; Muscle, Skeletal/metabolism* ; Muscle Development/genetics*

To study the role of BAMBI in adipogenesis, we constructed lentivirus interfering vector targeting on porcine BAMBI, packaged and infected the porcine preadipocyte. The differentiation state of preadipocyte was detected by Oil Red O staining and Oil Red O extraction assay and the expression levels of adipogenic marker genes were detected by Real-time qPCR and Werstern bloting. Results show that BAMBI expression was significant decreased after lentivirus infection, which was repressed more than 60% by shRNA2. Moreover, knockdown BAMBI increased the lipid accumulation of porcine preadipocyte and improved the expression of PPARγ (peroxisome proliferator-activated receptorγ) and ap2 (adipocyte protein 2). In summary, these data indicated that BAMBI inhibited adipocyte differentiation by facilitating the phosphorylation of ERK1/2.
Adipocytes ; cytology ; Adipogenesis ; Animals ; Cell Differentiation ; Membrane Proteins ; metabolism ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; PPAR gamma ; metabolism ; Phosphorylation ; Swine

To clarify the function and molecular mechanism of miR-155 in myogenic differentiation of C2C12, we constructed adenovirus over-expression vector of miR-155, then C2C12 cells were infected by adenovirus and induced myogenic differentiation. First, we observed the morphology of C2C12 after differentiation. Then the mRNA and protein expressions of myogenic markers (MyoD, MyoG and MyHC) were detected by qPCR and western blotting. Subsequently, the dual luciferase reporter gene assay was carried out to validate putative target gene (TCF4) of miR-155. Meanwhile, mRNA level of TCF4 was analyzed after over-expressing miR-155. The results show that over-expressed miR-155 reduced myotubes formation. Moreover, the mRNA and protein expression of MyoG and MyHC decreased significantly (P < 0.01). Further research demonstrated miR-155 bound the one (4532-4538) of three putative sites (1487-1493,1516-1522, 4532-4583) of TCF4 by luciferase reporter gene assay and the mRNA level of TCF4 decreased notably (P < 0.05). The data suggest that miR-155 inhibited myogenic differentiation of C2C12 through targeted TCF4.
Animals ; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors ; genetics ; Cell Differentiation ; Cell Line ; Genetic Vectors ; Mice ; MicroRNAs ; genetics ; Myoblasts ; cytology ; Myogenin ; genetics ; metabolism ; Myosin Heavy Chains ; genetics ; metabolism ; RNA, Messenger ; genetics ; Transcription Factor 4

Retinol-binding protein 4 (RBP4) is adipocyte-derived secreted adipokines and elevated RBP4 expression level was closely related to insulin resistance and type II diabetes mellitus. However, the exact mechanisms are unknown. To clarify the mechanism, RBP4 lentivirus particles were packaged to infect porcine preadipocytes. Then porcine preadipocytes were activated by insulin or induced model of insulin resistance. RBP4 interference efficiency and the gene expression of each treatment groups in PI3K/Akt pathways were examined by QRT-PCR and Western blotting. The result shows that RBP4 mRNA and protein expressions were suppressed more than 60% (P < 0.01). Furthermore, no matter under insulin stimulation or insulin resistance, RBP4 knockdown significantly increased the mRNA expressions of AKT2, PI3K, GLUT4 and IRS1 compared with the control. The protein phosphorylate levels of AKT2, PI3K, IRS1 arised, meanwhile enhanced the AKT2, PI3K, GLUT4 total protein expressions. Collectively, knockdown of RBP4 increased the insulin sensitivity through upregulated PI3K/Akt pathways related factors' expression and phosphorylation in porcine adipocytes. This research will provide a new idea to treat insulin resistance related diseases.
Adipocytes ; metabolism ; Animals ; Gene Knockdown Techniques ; Insulin Resistance ; physiology ; Phosphatidylinositol 3-Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Retinol-Binding Proteins, Plasma ; genetics ; pharmacology ; Signal Transduction ; Swine

To clarify the function of miR-103 in the differentiation of porcine preadipocyte, we carried out real-time PCR to detect the expression pattern of miR-103 during adipogenesis, and clarified its expression tendency through cell differentiation. Then we used adenovirus that overexpressed miR-103 to infect porcine preadipocyte. Subsequently, mRNA and protein expression of adipogenesis marker--PPARgamma and aP2 was analyzed by real-time PCR and Western blotting. At last, Oil-Red O staining was used to detect lipids accumulation in the 8th day after adipogenic inducement. The expression of miR-103 increased during adipocyte differentiation; compared with the control, the preadipocyte infected by pAd-miR-103 had an elevated expression level of adipocyte marker gene PPARgamma, aP2, and obvious lipid droplet was seen in the 8th day after adipogenic inducement. These results showed that miR-103 can enhance adipogenesis in primary cultured porcine adipocytes.
Adenoviridae ; genetics ; metabolism ; Adipocytes ; cytology ; metabolism ; Adipogenesis ; genetics ; Animals ; Base Sequence ; Cell Differentiation ; MicroRNAs ; genetics ; metabolism ; Molecular Sequence Data ; PPAR gamma ; genetics ; metabolism ; Primary Cell Culture ; RNA, Messenger ; genetics ; metabolism ; Swine ; Transfection

Estrogen-related receptor a (ERRalpha) is a key regulator for energy metabolism and adipogenesis. However, its role in lipolysis is unknown. To study the function of ERRalpha in lipolysis, primary cultured differentiated porcine adipocytes were treated by a specific inverse agonist XCT790 or infected with adenoviral vector expressed ERRalpha for 48 h, in the absence and/or presence of specific protein kinase A (PKA) inhibitor or extracellular signal-related kinase (ERK) inhibitor. Then, we measured the triglyceride (TG) content and the glycerol release into the culture media to analysis the effect of ERRalpha on lipolysis; Further, we analyzed the expression of PPARgamma, perilipin A, p-perilipin A, HSL and ATGL with Western blotting. Here, we found that ERRalpha significantly increased adipocytes differentiation, TG accumulation and glycerol release. Separately or simultaneously block the PKA and ERK pathway do not significantly altered the effect of ERRalpha on glycerol release. ERRalpha significantly up-regulated the proteins expression of PPARgamma, perilipin A, HSL and ATGL, while the p-perilipin A protein level was not significantly changed. These findings imply that ERRalpha could increase lipolysis via up-regulating HSL and ATGL, thereby to supply more FFA as substrate for a larger turnover of cellular triglyceride pool during adipocytes differentiation.
Adipocytes ; cytology ; metabolism ; Animals ; Animals, Newborn ; Cells, Cultured ; Glycerol ; analysis ; Lipase ; metabolism ; Lipolysis ; physiology ; Receptors, Estrogen ; metabolism ; physiology ; Sterol Esterase ; metabolism ; Swine ; Triglycerides ; analysis

MED1 is a key transcription co-activator subunit of the Mediator complex that is essential for RNA polymerase II-dependent transcription. MED1 functions as a co-activator for PPARs and other nuclear receptors and transcription factors, and plays an important role in lipid metabolism. To examine how MED1 might affect plasma lipids, plasma triglyceride, cholesterol levels, and lipoprotein profiles, were measured in MED1(deltaLiv) mice fasted for 24, 48 and 72 hours. Histological changes in liver sections from MED1(deltaLiv) mice after 72 hours of fasting were also examined using H&E staining. There was no fat accumulation in livers of MED1(deltaLiv) mice compared to MED1(fl/fl) and PPARalpha -/- control mice after 72 hours of fasting. Compared with MEDl(fl/fl) mice, plasma triglycerides in MED1(deltaLiv) mice were significantly increased after 24, 48 and 72 hours of fasting, and plasma cholesterol was significantly increased after 48 and 72 hours of fasting. Lipoprotein profiles were similar in fed MED1(fl/fl) and MED1(deltaLiv) mice. However, very low density lipoprotein (VLDL) was significantly increased in MED1(deltaLiv) mice after 24 hours of fasting. We conclude that, hyperlipidemia in MED1(deltaLiv) mice in response to fasting is due to the accumulation of VLDL, which suggests that MED1 plays a pivotal role in the regulation of plasma triglyceride and cholesterol levels.
Animals ; Cholesterol ; blood ; Fasting ; Hyperlipidemias ; blood ; Lipoproteins, VLDL ; blood ; Liver ; chemistry ; Mediator Complex Subunit 1 ; genetics ; physiology ; Mice ; Mice, Knockout ; Triglycerides ; blood

The Forkhead box O1 (FoxO1) transcription factor governs muscle growth, metabolism and cell differentiation. However, its role in myoblast differentiation is unclear. To study the biological function of FoxO1 during differentiation in porcine primary myoblast, we constructed stably FoxO1 over-expressed porcine myoblast mediated by liposome and adopted morphological observation, quantitative real-time RT-PCR and Western blotting methods to analyze FoxO1 and early and late myogenic regulation factors MyoD and myogenin expression. During differentiation the mRNA level of FoxO1 was significantly increased. However, the total protein did not change but the phosphorylation of FoxO1 was upregulated. Furthermore, overexpression of FoxO1 in porcine myoblast decreased MyoD and myogenin mRNA, whereas MyoD protein changed little and myogenin was significantly suppressed (P < 0.05). These results indicated that FoxO1 delays and negatively regulates the porcine myoblast differentiation. Moreover, FoxO1 may play a critical role in muscle fiber-type specification through the inhibition of myogenic regulation factors.
Animals ; Animals, Newborn ; Cell Differentiation ; genetics ; Cells, Cultured ; Forkhead Transcription Factors ; biosynthesis ; genetics ; Muscle, Skeletal ; cytology ; metabolism ; Myoblasts ; cytology ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Swine

We demonstrated the effect of resveratrol on porcine primary preadipocytes apoptosis, to study the intracellular molecular mechanism. Porcine primary preadipocyte was treated with different concentration of resveratrol (0 micromol/L, 50 micromol/L, 100 micromol/L, 200 micromol/L, 400 micromol/L). We used optical microscope and fluorescence microscope to observe morphological changes during apoptosis after Hoechst 33258 Fluorescent dyes staining; and RT-PCR and Western blotting to measure the expression of apoptosis-associated gene sirt1, caspase-3, bcl-2, bax, p53, NF-kappaB. Primary preadipocyte apoptosis was apparent, accompanied by reduced cell volume, chromatin condensation, and nuclear shrinkage. Compared to the control and low concentration group, high dose group (200 micromol/L) significantly increased the ratio of primary preadipocyte apoptosis. The expression of sirt1, caspase-3, and bax was up-regulated markedly in response to resveratrol; in contrast, apoptotic inhibitor bcl-2, p53, NF-kappaB down-regulated. We further proved fact that resveratrol can specifically promote the activity of sirt1; moreover, activated sirt1 modulates the activity of caspase-3 and bcl-2 family, involving in transcriptional regulation of p53 and NF-kappaB through antagonizing factor-induced acetylation. Taken together, our data established resveratrol as new regulator in porcine primary preadipocyte apoptosis via activating the expression of sirt1, modulating activity of apoptotic-associated factor.
Adipocytes ; cytology ; Adipogenesis ; Animals ; Antioxidants ; pharmacology ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cells, Cultured ; Sirtuin 1 ; metabolism ; Stilbenes ; pharmacology ; Swine