Refeeding and insulin activate the AKT/p70S6 kinase pathway without affecting IRS1 tyrosine phosphorylation in chicken muscle

p70 S6 kinase (p70S6K) is a key enzyme involved in the control of protein synthesis. We have previously shown that this kinase is insulin sensitive in chicken muscle despite a relative insulin resistance in the early steps of insulin receptor signaling in this tissue, particularly with no change in tyrosine phosphorylation of the insulin receptor substrate 1 (IRS1). The aim of the present study is to further study the p70S6K pathway in chicken muscle. By analyzing in silico several kinases involved in the protein kinase B (PKB also called AKT)/target of rapamycin (TOR)/p70S6K pathway in the chicken, we showed that the amino acid sequence of the proteins exhibited a very high identity with their homologs in mammalian species and Drosophila. We investigated the regulation of these kinases in vivo or in vitro. Refeeding and insulin treatment significantly (P<0.05) increased the phosphorylation and/or activity of kinases upstream of p70S6K such as AKT and TOR. Similarly, refeeding and insulin increased the phosphorylation of p70S6K on key residues (i.e. T389, T229 and T421/S424) and the phosphorylation of a p70S6K downstream target, the ribosomal protein S6 (by 3-10-fold, P<0.05). Interestingly, we also showed an increase in the phosphorylation level of IRS1 on S632/S635, sites involved in insulin resistance. In conclusion, the AKT/TOR/p70S6K pathway is activated by refeeding and insulin injection, which might negatively regulate IRS1 tyrosine phosphorylation. These results indicate some particularities of the insulin signaling in chicken muscle and suggest the involvement of p70S6K in these features.     

Regulation of protein metabolism by insulin: value of different approaches and animal models

Insulin induces protein accretion by stimulating protein synthesis and inhibiting proteolysis. However, the mechanisms of regulation of protein metabolism by insulin are complex and still not completely understood. The use of approaches combining hyperinsulinemic clamp and isotopic methods, or measurement of the activation of intracellular kinases involved in insulin signaling, in addition to the use of different animal models in a comparative physiology process, provide better understanding of the potential regulation of protein metabolism by insulin. Studies using the clamp technique in lactating goats have shown a clear inhibitory effect of insulin on proteolysis, with an interaction between the effects of insulin and amino acids. Such studies revealed that the insulin-inhibited proteolysis is improved in lactating goats, this adaptative process limiting the mobilization of body protein under the conditions of amino acid deficit which occurs during early lactation. Insulin signaling studies in growing chickens have also provided some interesting features of insulin regulation compared to mammals. Refeeding or insulin injection leads to the activation of the early steps of insulin receptor signaling in the liver but not in the muscle. Muscle p70 S6 kinase, a kinase involved in the insulin activation of protein synthesis, was found to be markedly activated in response to insulin and to refeeding, suggesting that other signaling pathways than those classically described in mammalian muscles may be involved in signal transduction. Finally, although the role of insulin has been doubtful and has long been considered to be minor in ruminants and in avian species, this hormone clearly regulates protein metabolism in both species.     

MAPK信号通路对脂肪细胞分化的调控

促分裂原活化的蛋白激酶(MAPK)通路主要包括胞外信号调控激酶(ERK)、p38MAPK和氨基末端蛋白激酶(JNK)三条途径,参与调节细胞增殖、分化、凋亡及细胞间的功能同步等过程,是细胞信号转导方面最为活跃的研究领域之一。研究显示MAPK也参与脂肪细胞的分化调节并发挥重要作用。ERK和p38MAPK信号通路对脂肪细胞分化的调节在不同的实验模型中表现为正调控和负调控两种不同形式;而另一成员JNK能使胰岛素受体底物1的丝氨酸发生磷酸化,进而干扰胰岛素信号,从而抑制骨髓间充质干细胞(BMSCs)的成脂分化,即对脂肪细胞分化发挥负调控作用。论文就MAPK信号通路在脂肪细胞分化中的功能进行综述,为脂类代谢性疾病的诊断和治疗提供参考。     

Possible involvement of phosphatidylinositol 3-kinase in the maintenance of metaphase II attest in porcine oocytes matured in vitro

It has been reported that phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB) pathway plays a crucial role in the meiotic resumption and progression to the metaphase II (MII) stage of oocytes. However, the role of this pathway in meiotic arrest at the MII stage (cytostatic activity) is not well understood. In this study the effect of a PI3K inhibitor, LY294002, on the MAPK and p34^cdc2 kinase activities of matured porcine oocytes was examined. After maturation culture, both the MAPK and p34^cdc2 kinase activities in the oocytes were gradually decreased in a time-dependent manner. Although 25 µmol/L LY294002 did not affect either the MAPK or p34^cdc2 kinase activities, 50 µmol/L LY294002 suppressed the PKB phosphorylation and slightly decreased MAPK activity, but not the p34^cdc2 kinase activity. Therefore the effect of 10 µmol/L Ca²⁺ ionophore which was reported as inducing a transient decrease of p34^cdc2 kinase but not MAPK activities, was also examined in LY294002-treated oocytes. By additional treatment with LY294002 after Ca²⁺ ionophore, both the MAPK and p34^cdc2 kinase activities were decreased in a time-dependent manner, concomitantly with improvement of pronuclear formation. Therefore, we concluded that PI3K is involved in the maintenance of MAPK activity in matured porcine oocytes.     

Ankyrin repeat and suppressor of cytokine signaling (SOCS) box-containing protein (ASB) 15 alters differentiation of mouse C2C12 myoblasts and phosphorylation of mitogen-activated protein kinase and Akt

Ankyrin repeat and suppressor of cytokine signaling box-containing protein (ASB) 15 is a novel ASB gene family member predominantly expressed in skeletal muscle. We have previously reported that overexpression of ASB15 delays differentiation and alters protein turnover in mouse C(2)C(12) myoblasts. However, the extent of ASB15 regulation of differentiation and molecular pathways underlying this activity are unknown. The extracellular signal-regulated kinase (Erk) 1/2 and phosphatidylinositol-3 kinase-Akt (PI3K/Akt; Akt is also known as protein kinase B) signaling pathways have a role in skeletal muscle growth. Activation (phosphorylation) of the Erk1/2 signaling pathway promotes proliferation, whereas activation of the PI3K/Akt signaling pathway promotes myoblast differentiation. Accordingly, we tested the hypothesis that ASB15 controls myoblast differentiation through its regulation of these kinases. Stably transfected myoblasts overexpressing ASB15 (ASB15+) demonstrated decreased differentiation, whereas attenuation of ASB15 expression (ASB15-) increased differentiation. However, ASB15+ cells had less abundance of the phosphorylated mitogen-activated protein kinase (active) form, despite decreased differentiation relative to control myoblasts (ASB15Con). The mitogen-activated protein kinase kinase inhibitor, U0126, effectively decreased mitogen-activated protein kinase phosphorylation and stimulated differentiation in ASB15- and ASB15Con cells. However, inhibition of the Erk1/2 pathway was unable to overcome the inhibitory effect of overexpressing ASB15 on differentiation (ASB15+), suggesting that the Erk1/2 pathway is likely not the predominant mediator of ASB15 activity on differentiation. Expression of ASB15 also altered phosphorylation of the PI3K/Akt pathway, as ASB15+ and ASB15- cells had decreased and increased Akt phosphorylation, respectively. These data were consistent with observed differences in differentiation. Administration of IGF-I, a PI3K/Akt activator, in ASB15+ was able to partially override the previously observed phenotype of delayed differentiation, whereas administration of the PI3K/ Akt inhibitor, LY294002, decreased phosphorylation of Akt and differentiation of all cell lines similar to the untreated ASB15+ myoblasts. These results provide initial evidence that ASB15 has a role in early myoblast differentiation and that its effects may be mediated in part by the PI3K/Akt signal transduction pathway.     

DUSP6通过负向调控ERK1/2通路抑制IBV的增值

为了阐明ERK(extracellular signal-regulated protein kinases)1/2通路在传染性支气管炎病毒(Infectious bronchitis virus,IBV)复制过程中的作用以及双特异性磷酸酶6(dual specificity phosphatase 6,DUSP6)对ERK的反馈性负向调控在IBV复制过程中的作用。本研究通过Western blot、Northern blot检测发现:IBV感染Vero和H1299细胞可导致ERK1/2的磷酸化水平和DUSP6表达均上调;利用MEK1/2特异性抑制剂U0126处理病毒感染的细胞后,可明显下调ERK1/2的磷酸化,同时抑制病毒的增殖;利用DUSP6的特异性抑制剂BCI抑制DUSP6的活性或者用siRNA阻断DUSP6的表达后,再感染IBV,发现ERK1/2的磷酸化水平增高,病毒蛋白的表达上调。综上,推测IBV感染细胞激活ERK1/2信号通路,有助于病毒的复制,同时,细胞通过诱导表达DUSP6,负向调控ERK1/2的磷酸化水平,抑制病毒增殖。     

Exposure to TNF-alpha but not IL-1beta impairs insulin-dependent phosphorylation of protein kinase B and p70S6k in mouse C2C12 myogenic cells

Tumor necrosis factor (TNF)-alpha is a proinflammatory cytokine considered to play an important role in muscle catabolism, but little is known about the mechanisms of its action. The aim of the present study was therefore to examine the effect of TNF-alpha pretreatment on glucose uptake and protein synthesis as well as the cellular content and phosphorylation of protein kinase B (PKB), p70S6k, Mitogen Activated Protein (MAP) kinase and p90rsk in mouse C2C12 myotubes stimulated with insulin. To determine whether interleukin (IL)-1beta might be involved in the catabolic action of TNF-alpha, the effects of IL-1beta were also tested. Experiments were performed on mouse C2C12 myoblasts subjected to differentiation in the presence of increasing concentrations of TNF-alpha (0.1-100 ng/ml) or IL-1 (5-50 ng/ml) for 5 or 6 days. Insulin (100 nmol/l) markedly stimulated glucose uptake in C2C12 myotubes (202.6% of control). This effect was profoundly attenuated by pretreatment with TNF-alpha at a concentration of 1 ng/ml (122.2% of control) and completely abolished by higher cytokine concentrations. Pretreatment of cells with TNF-alpha at a concentration of 1 ng/ml was also effective in diminishing the effect of insulin on protein synthesis, whereas higher cytokine concentrations prevented hormonal stimulation of protein synthesis in C2C12 myotubes. Pretreatment with TNF-alpha caused a significant decrease in PKB protein content. Insulin-mediated activation of protein kinase B was significantly diminished in cells differentiated in the presence of TNF-alpha. Treatment of C2C12 cells with insulin led to the gel mobility retardation of p70S6k indicating its phosphorylation and activation. In cells differentiated in the presence of TNF-alpha an approximately 2-fold decrease of insulin-mediated p70S6k phosphorylation was noted. Six-day differentiation of myogenic cells in the presence of TNF-alpha did not affect the protein content of p42MAPK, p44MAPK, p90rsk and phosphorylation of p42MAPK. Neither glucose uptake nor protein synthesis stimulated by insulin were affected significantly by pretreatment with IL-beta. Preincubation of myogenic cells with IL-1beta did not modify either the protein content of PKB and p70S6k or the insulin-stimulated phosphorylation of these kinases. In conclusion: i) high concentrations of TNF-alpha, but not IL-beta, present in the extracellular environment during myoblast differentiation prevent the stimulatory action of insulin on glucose uptake and protein synthesis; ii) insulin resistance induced by TNF-alpha in C2C12 myogenic cells could be associated with the decreased insulin-mediated phosphorylation of PKB and p70s6k, but not with the basal phosphorylation of p42MAPK.     

Vitamin E regulates bovine granulosa cell apoptosis via NRF2-mediated defence mechanism by activating PI3K/AKT and ERK1/2 signalling pathways

High-yield dairy cows are usually subject to high-intensive cell metabolism and produce excessive reactive oxygen species (ROS). Once ROS is beyond the threshold of scavenging ability, it can induce oxidative stress, imperilling the reproductive performance of cows. The study was to investigate the effects of vitamin E (VE) on H₂O₂-induced proliferation and apoptosis of bovine granulosa cells and the underlying molecular mechanism. Granulosa cells were pretreated with VE for 24 hr and then treated with H₂O₂ for 6 hr. The results showed that VE treatment decreased the intracellular ROS levels, increased the MDA content, and improved the antioxidant enzyme activity in a dose-dependent manner. Furthermore, VE treatment promoted the proliferation and inhibited apoptosis in granulosa cells by up-regulation of CCND1 and BCL2 levels and down-regulation of P21, BAX, and CASP3 levels. The cytoprotective effects of VE were attributed to the activation of the NRF2 signalling pathway. Knockdown of the NRF2 impaired the cytoprotective effects of VE on granulosa cells. Besides, the PI3K/AKT and ERK1/2, but not the p38 signalling pathway is involved in the regulation of VE-mediated cell proliferation and apoptosis. The PI3K/AKT inhibitor LY294002 and ERK1/2 inhibitor SCH772984 inhibited the VE-induced granulosa cell proliferation and promoted apoptosis, whereas the p38 inhibitor SB203580 had the opposite effects. These results were confirmed by proliferation and apoptosis-related gene expression at mRNA and protein levels. The results also showed that the PI3K/AKT inhibitor LY294002 and ERK1/2 inhibitor SCH772984 inhibited VE-induced NRF2, GCLC, GCLM, and HO-1 expression, whereas the p38 inhibitor SB203580 not. Overall, the results demonstrated that VE-regulated granulosa cell proliferation and apoptosis via NRF2-mediated defence system by activating the PI3K/AKT and ERK1/2 signalling pathway.     

表皮生长因子调控猪肠上皮细胞中钠依赖Ⅱb型磷转运蛋白表达的细胞信号通路研究

本研究旨在探讨表皮生长因子(EGF)调控猪小肠上皮细胞IPEC-J2中钠依赖Ⅱb型磷转运蛋白(NaPi-Ⅱb)表达的分子机制。试验分别用EGF受体酪氨酸激酶抑制剂(tyrphostin AG1478)、蛋白激酶A(PKA)抑制剂(H89)、蛋白激酶C(PKC)抑制剂(k4393)、p38抑制剂(SB203580)、细胞外信号调节激酶(ERK)抑制剂(PD98059)、c-Jun氨基末端激酶(JNK)抑制剂(anisomycin)与EGF共同处理IPEC-J2细胞,利用Western blot检测相关通路蛋白及目的蛋白(NaPi-Ⅱb)的表达水平。结果显示:相较于对照组,EGF处理后NaPi-Ⅱb表达水平显著降低(P0.05);相较于无抑制剂组,EGF受体、PKA、PKC、丝裂原活化蛋白激酶(MAPK)/p38、MAPK/ERK1/2、MAPK/JNK的特异性抑制剂处理IPEC-J2后,NaPi-Ⅱb表达水平显著提高(P0.05),其中添加MAPK/ERK1/2特异性抑制剂显著降低了MAPK/ERK1/2在Tyr204位点的磷酸化水平(P0.05),添加MAPK/JNK的特异性抑制剂显著降低了MAPK/JNK1/2/3在Thr183和Tyr185位点的磷酸化水平(P0.05),说明该2组抑制剂对该通路的抑制作用是通过降低上述位点的磷酸化水平实现的。本研究结果表明EGF受体、PKA、PKC、p38、ERK和JNK均介导了EGF调控IPEC-J2细胞中NaPi-Ⅱb的表达。     

Early steps of insulin receptor signaling in chicken and rat: apparent refractoriness in chicken muscle

The early steps of insulin receptor (IR) signaling (tyrosine phosphorylation of IR beta-subunit, IRS-1 and Shc and PI 3'-kinase activity) have been characterized in two target tissues in the chicken: liver and muscle. The signaling cascade appeared to depend on nutritional status in the liver, but not in muscle (with a possible exception for a minor tyrosine phosphorylation of the 52 kDa Shc isoform). In this study, we compared the responses of the liver and muscle to exogenous insulin (10 or 1000 mU/kg) in chickens and rats. In the liver, IRS-1 and Shc proteins were present in smaller amounts and the regulatory subunit p85 of PI 3'-kinase was present in larger amounts in chickens than in rats. In the basal state (saline injection), the level of tyrosine phosphorylation of IR was lower, and that of Shc higher, in chickens than in rats. PI 3'-kinase activity in chickens was half that in rats. Insulin activated all components of the cascade in a dose-dependent manner in both species. A different pattern was observed in the muscle. In the basal state, the levels of tyrosine phosphorylation of IR and of PI 3'-kinase activity were much higher in chickens than in rats (by factors of 2 and 30, respectively). Insulin strongly activated all components of the cascade in rats (but with no significant increase in the phosphorylation of Shc). No activation was observed in chickens (with only a slight but significant increase in the tyrosine phosphorylation of Shc). The insulin cascade therefore appears to respond normally in chicken liver but to be refractory in chicken muscle. The large amount of p85 and high levels of PI 3'-kinase activity in muscle may contribute to this situation, making chicken muscle an interesting model of insulin resistance.     

猪瘟病毒非结构蛋白NS5A与Beclin1相互作用并促进病毒增殖

为探究宿主蛋白Beclin1在猪瘟病毒(classical swine fever virus, CSFV)非结构蛋白NS5A激活细胞自噬反应过程中的作用及具体分子机制,本研究在感染CSFV及表达NS5A蛋白的ST细胞中,利用qRT-PCR方法检测Beclin1、PI3K/Akt通路相关因子表达变化情况;利用激光共聚焦、Co-IP及GST-pulldown等方法研究Beclin1与NS5A相互作用关系;通过在ST细胞中过表达或敲低Beclin1,研究其对CSFV复制的影响。结果表明,ST细胞感染猪瘟病毒或外源表达NS5A蛋白,Beclin1转录和蛋白表达水平均显著升高,且PI3K/Akt通路相关因子表达水平与之呈正相关。此外,CSFV NS5A蛋白与Beclin1蛋白在细胞中存在共定位且具有相互作用。最后,作者发现在细胞中过表达Beclin1,对CSFV复制起到明显促进作用;反之,利用siRNA敲低Beclin1后,抑制PI3K/Akt通路活化,CSFV增殖表现出明显抑制效应。以上结果表明,Beclin1蛋白对CSFV复制具有促进作用,其机制是通过与NS5A的相互作用调控PI3K/Ak...     

Changes of insulin-mediated protein kinases phosphorylation and the expression of IGFBP-3 in skeletal muscle of streptozotocin-diabetic mice

The purpose of the present study was to investigate potential changes in expression and activation of Ser/Thr protein kinases as well as in the level of insulin-like growth factor-binding proteins (IGFBPs) in skeletal muscle of streptozotocin (STZ)-diabetic mice. We have examined the basal and insulin-mediated phosphorylation of protein kinase B (PKB), protein kinase Czeta (PKCzeta), p70(S6k), mitogen-activated protein kinase (MAPK)/p90(rsk) pathway and the expression of IGFBP-3, -4, and -5 in mice selected for body weight gain (line C) and reduction (line L). Apart from IGFBP-3 level, which was higher in C line, the diabetes-associated changes in signaling components examined in present work were similar in both lines of mice. The expression of PKB in skeletal muscle was similar in control and diabetic mice. Insulin increased the Ser473 phosphorylation of PKB in both experimental groups however, in diabetic mice the insulin-dependent PKB phosphorylation was more evident in comparison to control group. Neither protein level nor insulin-stimulated p70(S6k) activation were modified by STZ-diabetes. Basal PKC phosphorylation was augmented in muscle of diabetic mice and it was not increased following insulin injection. No apparent differences in levels of p42(MAPK), p44(MAPK) and p90(rsk) protein in gastrocnemius muscles between control and STZ-treated mice were observed. Basal phosphorylation of p90(rsk) in diabetic mice was markedly elevated in comparison to the control. In muscle of C-line mice, insulin stimulated the p90(rsk) activity to the same extent in both experimental groups (+22% over appropriate basal value). Insulin-mediated stimulation of p90(rsk) in muscle of L-line mice amounted to +26% and +14%, for control and diabetic mice, respectively. Protein level of IGFBP-3 in muscle of diabetic C-line mice was augmented by approx. 28% when compared to the control, whereas the expression of IGFBP-4 and -5 was not modified by STZ-diabetes. In conclusion: diabetes-associated changes in the insulin signaling in skeletal muscle involve: 1) enhanced insulin-dependent phosphorylation of PKB; 2) increased basal phosphorylation of PKC and its resistance to stimulatory action of insulin; 3) increased basal phopshorylation of p90(rsk), and 4) augmented IGFBP-3 protein level, which can potentially contribute to disruption of anabolic signals in this tissue.     

厚朴酚在体外对LPS诱导细胞因子释放及ERK通路激活的影响

利用MTT方法检测厚朴酚细胞毒性，ELISA方法检测厚朴酚对LPS介导炎症中IL-1、IL-6、IL-10释放的调节作用，Western—blot方法检测对ERK1／2活化的调节作用。结果显示，LPS处理过的RAW264．7细胞在经过不同剂量厚朴酚处理后，其IL-1、IL-6的释放随厚朴酚剂量的增加在减少，而IL-10则随药物剂量增加而增加；厚朴酚对LPS介导的ERK／MAPK通路激活作用显示一定的抑制作用。结果表明，在一定剂量范围内厚朴酚可以缓解LPS刺激的细胞因子的释放，在炎症过程中可以通过抑制ERK／MAPK活化来抑制炎症发生。     

PS48 promotes in vitro maturation and developmental competence of porcine oocytes through activating PI3K/Akt signalling pathway

Oocyte maturation plays a vitally important role in porcine reproduction. Regrettably, the quality of oocytes matured in vitro is weaker than that of in vivo matured oocytes. We collected and cultivated porcine cumulus oocyte complexes (COCs) in vitro with phosphoinositide-dependent kinase 1 (PDK1) activator 5-(4-chloro-phenyl)-3-phenyl-pent-2-enoic acid (PS48), whose concentrations were 0, 2, 5, 10 and 20 µM to investigate whether the phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) signalling pathway would impact the oocyte quality. The results showed that 10 µM PS48 increased the oocyte proportion of metaphase II (MII) stage and improved the expansion of cumulus cells (CCs). What's more, the activation of PI3K/Akt signalling pathway could regulate the expression of maturation-related genes and proteins. The results of quantitative real-time PCR showed that 10 µM PS48 increased the mRNA and protein levels of Akt and regulated maturation-related genes, including cyclin B1, MOS, BMP15, GDF9, CDC2, mTOR, BAX, BCL2 and caspase-3. The results of Western blot indicated that 10µM PS48 increased the protein abundance of Akt, phosphorylation of Akt Thr308 (p-Akt^Thr308) and cyclin B1, but decreased the protein abundance of pro-apoptotic BAX. These results suggested that adding 10 µM PS48 to mature culture medium could promote the maturation of porcine oocytes, potentially through activating the PI3K/Akt signalling pathway.     

Effect of porcine glucagon‐like peptides‐2 on tight junction in GLP‐2R + IPEC‐J2 cell through the PI3k/Akt/mTOR/p70S6K signalling pathway

Because of rare glucagon‐like peptide‐2 (GLP‐2) receptor (+) cells within the gut mucosa, the molecular mechanisms transducing the diverse actions of GLP‐2 remain largely obscure. This research identified the naturally occurring intestinal cell lines that endogenously express GLP‐2R and determined the molecular mechanisms of the protective effects of GLP‐2‐mediated tight junctions (TJ) in GLP‐2R (+) cell line. (i) Immunohistochemistry results showed that GLP‐2R is localised to the epithelia, laminae propriae and muscle layers of the small and large bowels of newborn piglets. (ii) GLP‐2R expression was apparent in the cytoplasm of endocrine cells in IPEC‐J2 cell lines. (iii) The protein expressions of ZO‐1, claudin‐1, occludin, p‐PI₃K, p‐Akt, p‐mTOR and p‐p70^S6K significantly (p < 0.05) increased in GLP‐2‐treated IPEC‐J2 cells, and all of them significantly (p < 0.05) decreased when LY‐294002 or rapamycin was added. GLP‐2 improves intestinal TJ expression of GLP‐2R (+) cells through the PI₃k/Akt/mTOR/p70^S6K signalling pathway.     

Differential regulation of protein synthesis in skeletal muscle and liver of neonatal pigs by leucine through an mTORC1-dependent pathway

Neonatal growth is characterized by a high protein synthesis rate that is largely due to an enhanced sensitivity to the postprandial rise in insulin and amino acids, especially leucine. The mechanism of leucine’s action in vivo is not well understood. In this study, we investigated the effect of leucine infusion on protein synthesis in skeletal muscle and liver of neonatal pigs. To evaluate the mode of action of leucine, we used rapamycin, an inhibitor of mammalian target of rapamycin (mTOR) complex-1 (mTORC1). Overnight-fasted 7-day-old piglets were treated with rapamycin for 1 hour and then infused with leucine (400 μmol·kg -1 ·h -1 ) for 1 hour. Leucine infusion increased the rate of protein synthesis, and ribosomal protein S6 kinase 1 (S6K1) and eukaryotic initiation factor (eIF) 4E-binding protein-1 (4E-BP1) phosphorylation in gastrocnemius and masseter muscles (P < 0.05), but not in the liver. The leucine-induced stimulation of protein synthesis and S6K1 and 4E-BP1 phosphorylation were completely blocked by rapamycin, suggesting that leucine action is by an mTORC1-dependent mechanism. Neither leucine nor rapamycin had any effect on the activation of the upstream mTORC1 regulators, AMP-activated protein kinase and protein kinase B, in skeletal muscle or liver. The activation of eIF2a and elongation factor 2 was not affected by leucine or rapamycin, indicating that these two pathways are not limiting steps of leucine-induced protein synthesis. These results suggest that leucine stimulates muscle protein synthesis in neonatal pigs by inducing the activation of mTORC1 and its downstream pathway leading to mRNA translation.     

p38丝裂原活化蛋白激酶信号途径调节骨骼肌生长发育的机理

p38丝裂原活化蛋白激酶(MAPK)信号途径是成肌细胞分化过程中重要的调节途径。p38 M APK蛋白在成肌细胞分化过程中受上游丝裂原活化蛋白激酶激酶3(M KK3)、丝裂原活化蛋白激酶激酶6(MKK6)的激活而活化其下游成肌分化蛋白(MyoD)、肌生成素5(Myf5)、肌形成蛋白(myogenin)、肌肉调节因子4(MRF4)等肌肉调节因子。p38 MAPK信号途径的活化可以进一步增加骨骼肌纤维细胞蛋白含量,增加肌纤维长度和横截面直径,使骨骼肌纤维在数量不变的前提下质量大幅度提高,本文就p38 MAPK信号途径调节骨骼肌生长发育的机理进行综述。     

Biphasic activation of PI3K/Akt and MAPK/Erk1/2 signaling pathways in bovine herpesvirus type 1 infection of MDBK cells

ABSTRACT: Many viruses have been known to control key cellular signaling pathways to facilitate the virus infection. The possible involvement of signaling pathways in bovine herpesvirus type 1 (BoHV-1) infection is unknown. This study indicated that infection of MDBK cells with BoHV-1 induced an early-stage transient and a late-stage sustained activation of both phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen activated protein kinases/extracellular signal-regulated kinase 1/2 (MAPK/Erk1/2) signaling pathways. Analysis with the stimulation of UV-irradiated virus indicated that the virus binding and/or entry process was enough to trigger the early phase activations, while the late phase activations were viral protein expression dependent. Biphasic activation of both pathways was suppressed by the selective inhibitor, Ly294002 for PI3K and U0126 for MAPK kinase (MEK1/2), respectively. Furthermore, treatment of MDBK cells with Ly294002 caused a 1.5-log reduction in virus titer, while U0126 had little effect on the virus production. In addition, the inhibition effect of Ly294002 mainly occurred at the post-entry stage of the virus replication cycle. This revealed for the first time that BoHV-1 actively induced both PI3K/Akt and MAPK/Erk1/2 signaling pathways, and the activation of PI3K was important for fully efficient replication, especially for the post-entry stage.