Myostatin regulates preadipocyte differentiation and lipid metabolism of adipocyte via ERK1/2

Myostatin is known as an inhibitor of muscle development, but its role in adipogenesis and lipid metabolism is still unclear, especially the underlying mechanisms. Here, we demonstrated that myostatin inhibited 3T3-L1 preadipocyte differentiation into adipocyte by suppressing C/EBPα (CCAAT/enhancer-binding protein α) and PPARγ (peroxisome-proliferator-activated receptor γ), also activated ERK1/2 (extracellular-signal-regulated kinase 1/2). Furthermore, myostatin enhanced the phosphorylation of HSL (hormone-sensitive lipase) and ACC (acetyl-CoA carboxylase) in fully differentiated adipocytes, as well as ERK1/2. Besides, we noted that myostatin markedly raised the levels of leptin and adiponectin release and mRNA expression during preadipocyte differentiation, but the levels were inhibited by myostatin treatments in fully differentiated adipocytes. These results suggested that myostatin suppressed 3T3-L1 preadipocyte differentiation and regulated lipid metabolism of mature adipocyte, in part, via activation of ERK1/2 signalling pathway.     

Role of C/EBPβ-LAP and C/EBPβ-LIP in early adipogenic differentiation of human white adipose-derived progenitors and at later stages in immature adipocytes

We investigated the role of the major isoforms of CCAAT enhancer binding protein β (C/EBPβ), C/EBPβ-LAP and C/EBPβ-LIP, in adipogenesis of human white adipose-derived stromal/progenitor cells (ASC). C/EBPβ gene expression was transiently induced early in adipogenesis. At later stages, in immature adipocytes, the C/EBPβ mRNA and protein levels declined. The C/EBPβ-LIP protein steady-state level decreased considerably stronger than the C/EBPβ-LAP level and the C/EBPβ-LIP half-life was significantly shorter than the C/EBPβ-LAP half-life. The turn-over of both C/EBPβ-isoforms was regulated by ubiquitin/proteasome-dependent degradation. These data suggest that the protein stability of the C/EBPβ-isoforms is differentially regulated in the course of adipogenesis and in immature adipocytes. Constitutive overexpression of C/EBPβ-LIP had antiadipogenic activity in human ASC. C/EBPβ-LAP, which promotes adipogenesis in mouse 3T3-L1 preadipocytes by directly activating expression of the adipogenic keyregulator PPARγ2, induced the expression of PPARγ2 and of the adipocyte differentiation gene product FABP4 in confluent ASC in the absence of adipogenic hormones. At later stages after hormone cocktail-induced adipogenesis, in immature adipocytes, constitutive overexpression of C/EBPβ-LAP led to reduced expression of PPARγ2 and FABP4, C/EBPα expression was downregulated and the expression of the adipocyte differentiation gene products adiponectin and leptin was impaired. These findings suggest that constitutive overexpression of C/EBPβ-LAP induces adipogenesis in human ASC and negatively regulates the expression of adipogenic regulators and certain adipocyte differentiation gene products in immature adipocytes. We conclude the regulation of both C/EBPβ gene expression and C/EBPβ-LIP and C/EBPβ-LAP protein turn-over plays an important role for the expression of adipogenic regulators and/or adipocyte differentiation genes in early adipogenic differentiation of human ASC and at later stages in human immature adipocytes.     

Myogenic gene expression at rest and after a bout of resistance exercise in young (18-30 yr) and old (80-89 yr) women.

The purpose of this study was to investigate mRNA expression of several key skeletal muscle myogenic controllers; myogenic differentiation factor (MyoD), muscle regulatory factor 4 (MRF4), myogenic factor 5 (Myf5), myogenin, myostatin, and myocyte enhancer factor 2 (MEF2) at rest and 4 h after a single bout of resistance exercise (RE) in young and old women. Eight young women (YW; 23 +/- 2 yr, 67 +/- 5 kg) and six old women (OW; 85 +/- 1 yr, 67 +/- 4 kg) performed 3 sets of 10 repetitions of bilateral knee extensions at 70% of one repetition maximum. Muscle biopsies were taken from the vastus lateralis before and 4 h after RE. Using real-time RT PCR, mRNA from the muscle samples was amplified and normalized to GAPDH. At rest, OW expressed higher (P < 0.05) levels of MyoD, MRF4, Myf5, myogenin, and myostatin compared with YW. In response to RE, there was a main time effect (P < 0.05) for the YW and OW combined in the upregulation of MyoD (2.0-fold) and MRF4 (1.4-fold) and in the downregulation of myostatin (2.2-fold). There was a trend (P = 0.08) for time x age interaction in MRF4. These data show that old women express higher myogenic mRNA levels at rest. The higher resting myogenic mRNA levels in old women may reflect an attempt to preserve muscle mass and function. When challenged with RE, old women appear to respond in a similar manner as young women.     

Contrary Effects of BMP-2 and ATRA on Adipogenesis in Mouse Mesenchymal Fibroblasts

This study evaluates the effects of bone morphogenetic protein 2 (BMP-2) and all trans retinoic acid (ATRA) on adipogenesis in primary mouse embryo fibroblasts (MEFs). In BMP-2-treated MEFs, lipid accumulation and substantial induction of the adipocyte specific marker 442-aP2 suggested the conversion of MEFs into adipocytes. Such adipogenesis was found to be mediated through sequential induction of C/EBPα, C/EBPβ, and PPARγ. Both the BMP/Smad and BMP/p38 pathways contributed to the adipocyte differentiation. Contrary to the effects of BMP-2, ATRA was demonstrated to inhibit adipocyte differentiation in MEFs. Semi-quantitative RT-PCR analysis revealed that ATRA caused a selective inhibition of both the basal and induction levels of C/EBPα and PPARγ, without altering the expression pattern of C/EBPβ. Taken together, these data suggest the roles of BMP-2 and ATRA in adipogenic differentiation of primary MEFs, and the possible molecular mechanism that involves the regulation of C/EBPα, C/EBPβ, and PPARγ.     

Quercetin glycosides prevent dexamethasone-induced muscle atrophy in mice

Although quercetin has numerous biological benefits, including preventing muscle atrophy due to disuse, no reports have been published to date about the preventive effects and molecular mechanisms underlying drug-induced muscle atrophy. Highly soluble and bioavailable quercetin glycosides (QGs) were used to examine the inhibition of dexamethasone (DEX)-induced muscle atrophy in vivo. Male BALB/cCrSlc mice were treated with or without QGs for 7 days ad libitum, followed by addition of DEX to their drinking water for a further 7 days. The weight of gastrocnemius (GM) adjusted by body weight was significantly decreased on day 7 after DEX treatment. DEX-induced decrease of GM weight was improved by QG co-administration on day 7. The mRNA levels of muscle atrophy-related genes in the gastrocnemius were significantly lowered by QGs on day 1. In particular, the expression of myostatin, a master regulator of muscle mass homeostasis, was suppressed to that of the control level. In murine C2C12 myotubes, quercetin elevated the phosphorylation of Akt, which are downstream of the myostatin pathway, as well as expression of atrogenes. We demonstrated the protective effect of QGs in DEX-induced muscle atrophy, which might depend on the suppression of myostatin signaling.     

Stable suppression of myostatin gene expression in goat fetal fibroblast cells by lentiviral vector‐mediated RNAi

Myostatin (MSTN) is a secreted growth factor that negatively regulates skeletal muscle mass, and therefore, strategies to block myostatin‐signaling pathway have been extensively pursued to increase the muscle mass in livestock. Here, we report a lentiviral vector‐based delivery of shRNA to disrupt myostatin expression into goat fetal fibroblasts (GFFs) that were commonly used as karyoplast donors in somatic‐cell nuclear transfer (SCNT) studies. Sh‐RNA positive cells were screened by puromycin selection. Using real‐time polymerase chain reaction (PCR), we demonstrated efficient knockdown of endogenous myostatin mRNA with 64% down‐regulation in sh2 shRNA‐treated GFF cells compared to GFF cells treated by control lentivirus without shRNA. Moreover, we have also demonstrated both the induction of interferon response and the expression of genes regulating myogenesis in GFF cells. The results indicate that myostatin‐targeting siRNA produced endogenously could efficiently down‐regulate myostatin expression. Therefore, targeted knockdown of the MSTN gene using lentivirus‐mediated shRNA transgenics would facilitate customized cell engineering, allowing potential use in the establishment of stable cell lines to produce genetically engineered animals. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:452–459, 2015     

Expression of basic fibroblast growth factor results in the decrease of myostatin mRNA in murine C2C12 myoblasts

During the development and regeneration of skeletal muscle,many growth factors,such asbasic fibroblast growth factor (bFGF,FGF-2) and myostatin,have been shown to play regulating roles.bFGF contributes to promote proliferation and to inhibit differentiation of skeletal muscle,whereas myostatinplays a series of contrasting roles.In order to elucidate whether the expression of bFGF has any relationshipwith the expression of myostatin in skeletal muscle cells,we constructed a eukaryotic expression vector forthe expression of exogenous bFGF in murine C2C12 myoblasts.Quantitative RT-PCR assays indicated thatwith the increase of the expression of exogenous bFGF gene,the expression of endogenous myostatin genewas suppressed at mRNA level and protein level.     

Sleeping Beauty-mediated knockdown of sheep myostatin by RNA interference

Myostatin is a negative regulator of skeletal muscle growth. Myostatin dysfunction therefore offers a strategy for promoting animal muscle growth in livestock production. Knockdown of myostatin was achieved by combining RNA interference and the Sleeping Beauty (SB) transposon system in sheep cells. Four targeting sites of sheep myostatin were designed and measured for myostatin silencing in sheep fetal fibroblasts by real-time PCR. The sh3 construct induced significant decrease of myostatin gene expression by 90% (P < 0.05). Myostatin silencing induced by SB-mediated sh3 was further tested in stably transfected cells. SB transposition increased the integration frequency of genes into sheep genomes and mediated a more efficient myostatin knockdown than random integration of sh3. We suggest that SB-mediated shRNA provides a novel potential tool for gene knockdown in the donor cells of animal cloning.     

Nucleoside diphosphate kinase/Nm23 and Epstein–Barr virus

Compelling evidence indicates the pro-fibrogenic action of leptin in liver. Peroxisome proliferator-activated receptor-γ (PPARγ) can reverse hepatic stellate cell (HSC) activation and maintain HSC quiescence. HSC activation, a key step in the development of liver fibrosis, is coupled with the up-expression of leptin and the dramatic down-expression of PPARγ. The present study is aimed to assess the effect of leptin on PPARγ gene expression in primary cultured rat HSCs and investigate the related mechanisms by using Western blotting analysis, real-time PCR, transient transfection approach, and cell growth analysis. The results suggest that leptin negatively regulates PPARγ gene expression at mRNA level, protein level and PPARγ gene promoter activity level in HSCs. The inhibitory effect of leptin on PPARγ gene expression contributes to cell growth of activated HSCs in vitro. Phosphatidylinositol 3-kinase/AKT (PI-3 K/AKT) and extracellular signal-regulated kinase (ERK) signaling pathways mediate the leptin-induced inhibition of PPARγ gene expression. In summary, these findings suggest that leptin down-regulates PPARγ gene expression through activation of PI-3 K/AKT or ERK signaling pathway in primary cultured rat HSCs. Our results might provide novel insights into the mechanisms for the pro-fibrogenic action of leptin in liver.     

Ankrd26 gene disruption enhances adipogenesis of mouse embryonic fibroblasts

We previously reported that partial disruption of the Ankrd26 gene in mice leads to hyperphagia and leptin-resistant obesity. To determine whether the Ankrd26 mutation can affect the development of adipocytes, we studied mouse embryo fibroblasts (MEFs) from the mutant mice. We found that Ankrd26(-/-) MEFs have a higher rate of spontaneous adipogenesis than normal MEFs and that adipocyte formation is greatly increased when the cells are induced with troglitazone alone or with a mixture of troglitazone, insulin, dexamethasone, and methylisobutylxanthine. Increased adipogenesis was detected as an increase in lipid droplet formation and in the expression of several markers of adipogenesis. There was an increase in expression of early stage adipogenesis genes such as Krox20, KLF5, C/EBPβ, C/EBPδ, and late stage adipogenesis regulators KLF15, C/EBPα, PPARγ, and aP2. There was also an increase in adipocyte stem cell markers CD34 and Sca-1 and preadipocyte markers Gata2 and Pref-1, indicating an increase in both stem cells and progenitor cells in the mutant MEFs. Furthermore, ERK was found constitutively activated in Anrd26(-/-) MEFs, and the addition of MEK inhibitors to mutant cells blocked ERK activation, decreased adipogenesis induction, and significantly reduced expression of C/EBPδ, KLF15, PPARγ2, CD34, and Pref-1 genes. We conclude that Ankrd26 gene disruption promotes adipocyte differentiation at both the progenitor commitment and differentiation steps and that ERK activation plays a role in this process.     

绵羊肌肉H-FABP和PPARγ基因表达的发育性变化及其对肌内脂肪含量的影响

选取不同日龄的雄性哈萨克羊和新疆细毛羊,屠宰后取背最长肌,用索氏抽提法检测肌内脂肪（Intamuscular fat,IMF）含量,用荧光实时定量PCR法检测心脏脂肪酸结合蛋白（heart fatty acid-binding protein,H-FABP）和过氧化物酶体增殖物激活受体γ（peroxisome proliferator-activated receptorγ,PPARγ）基因表达的发育性变化极其对IMF含量的影响.结果表明：（1）随着日龄的增加,IMF含量在雄性哈萨克羊中持续上升,且在各生长期间的差异显著（P＜0.05）,而在新疆细毛羊的各生长期间无显著差异（P＞0.05）.雄性哈萨克羊的IMF含量在30～90日龄间极显著高于新疆细毛羊（P＜0.01）;（2）雄性哈萨克羊肌肉H-FABP基因的表达量在2日龄时最高,30日龄时降到最低,然后持续上升,各日龄间差异显著（P＜0.05）.新疆细毛羊中的表达量在2日龄时也最高,然后持续下降,到90日龄时降到了最低点,然后上升,各日龄间差异也显著（P＜0.05）;（3）雄性哈萨克羊肌肉PPARγ基因的表达量随着日龄的增加而呈下降趋势,各日龄间差异显著（P＜0.05）;新疆细毛羊中的表达量在2～60日龄期间持续上升且各日龄间差异显著（P＜0.05）,但在90日龄时降到最低点,然后上升;（4）在哈萨克羊中,H-FABP基因在30～90日龄间的表达量与IMF含量的相关系数为0.737（P＜0.01）,而PPARγ基因在2-90日龄间的表达量与之的相关系数为-0.835（P＜0.01）.