Hedgehog信号通路与动物骨骼肌发育调控

Hedgehog信号通路在动物胚胎期及出生后骨骼肌的生长发育过程中发挥着重要作用。本文综述了Hedgehog信号通路对骨骼肌细胞增殖分化及肌纤维特性的调控作用及其在骨骼肌发育过程中与其它信号通路交互作用最新研究进展,为畜禽肉品质改良和肌肉相关疾病治疗提供理论基础。     

调控骨骼肌纤维类型转化的信号通路

骨骼肌由异质性的肌纤维组成,不同类型的肌纤维具有不同的形态、代谢、生理和生化特性.根据不同肌纤维中表达的特异肌球蛋白重链亚型可将成体哺乳动物骨骼肌纤维分为4类,即Ⅰ,Ⅱa,Ⅱx和Ⅱb型.骨骼肌保持高度可塑性,当机体受到某些生理或病理刺激时,骨骼肌为了适应需要,通过激活胞内相关信号通路改变肌纤维特异基因的表达从而诱发肌纤维类型的转化.本文综述了细胞内参与调控肌纤维类型转化的多条重要信号通路,如Ca2+信号通路,Ras/MAPK信号通路及多种转录调节因子,辅激活因子和抑制子等,为改善肉类品质,提高运动训练效果及治疗肌肉相关疾病奠定了理论基础.     

Urotensin II Inhibits Skeletal Muscle Glucose Transport Signaling Pathways via the NADPH Oxidase Pathway

Our previous studies have demonstrated that the urotensin (UII) and its receptor are up-regulated in the skeletal muscle of mice with type II diabetes mellitus (T2DM), but the significance of UII in skeletal muscle insulin resistance remains unknown. The purpose of this study was to investigate the effect of UII on NADPH oxidase and glucose transport signaling pathways in the skeletal muscle of mice with T2DM and in C2C12 mouse myotube cells. KK/upj-AY/J mice (KK) mice were divided into the following groups: KK group, with saline treatment for 2 weeks; KK+ urantide group, with daily 30 µg/kg body weight injections over the same time period of urantide, a potent urotensin II antagonist peptide; Non-diabetic C57BL/6J mice were used as normal controls. After urantide treatment, mice were subjected to an intraperitoneal glucose tolerance test, in addition to measurements of the levels of ROS, NADPH oxidase and the phosphorylated AKT, PKC and ERK. C2C12 cells were incubated with serum-free DMEM for 24 hours before conducting the experiments, and then administrated with 100 nM UII for 2 hours or 24 hours. Urantide treatment improved glucose tolerance, decreased the translocation of the NADPH subunits p40-phox and p47-phox, and increased levels of the phosphorylated PKC, AKT and ERK. In contrast, UII treatment increased ROS production and p47-phox and p67-phox translocation, and decreased the phosphorylated AKT, ERK1/2 and p38MAPK; Apocynin abrogated this effect. In conclusion, UII increased ROS production by NADPH oxidase, leading to the inhibition of signaling pathways involving glucose transport, such as AKT/PKC/ERK. Our data imply a role for UII at the molecular level in glucose homeostasis, and possibly in skeletal muscle insulin resistance in T2DM.     

Autophagy Signaling in Skeletal Muscle of Infarcted Rats

Background

Heart failure (HF)-induced skeletal muscle atrophy is often associated to exercise intolerance and poor prognosis. Better understanding of the molecular mechanisms underlying HF-induced muscle atrophy may contribute to the development of pharmacological strategies to prevent or treat such condition. It has been shown that autophagy-lysosome system is an important mechanism for maintenance of muscle mass. However, its role in HF-induced myopathy has not been addressed yet. Therefore, the aim of the present study was to evaluate autophagy signaling in myocardial infarction (MI)-induced muscle atrophy in rats.

Methods/Principal Findings

Wistar rats underwent MI or Sham surgeries, and after 12 weeks were submitted to echocardiography, exercise tolerance and histology evaluations. Cathepsin L activity and expression of autophagy-related genes and proteins were assessed in soleus and plantaris muscles by fluorimetric assay, qRT-PCR and immunoblotting, respectively. MI rats displayed exercise intolerance, left ventricular dysfunction and dilation, thereby suggesting the presence of HF. The key findings of the present study were: a) upregulation of autophagy-related genes (GABARAPL1, ATG7, BNIP3, CTSL1 and LAMP2) was observed only in plantaris while muscle atrophy was observed in both soleus and plantaris muscles, and b) Cathepsin L activity, Bnip3 and Fis1 protein levels, and levels of lipid hydroperoxides were increased specifically in plantaris muscle of MI rats.

Conclusions

Altogether our results provide evidence for autophagy signaling regulation in HF-induced plantaris atrophy but not soleus atrophy. Therefore, autophagy-lysosome system is differentially regulated in atrophic muscles comprising different fiber-types and metabolic characteristics.     

Effect of Supplemental Dietary Zinc on the Mammalian Target of Rapamycin (mTOR) Signaling Pathway in Skeletal Muscle and Liver from Post-absorptive Mice

Zinc (Zn) is an essential trace element that functions in cellular signaling. The mammalian target of rapamycin (mTOR) regulates the initiation of protein synthesis. The objective of this study was to determine whether Zn could stimulate protein phosphorylation in the mTOR pathway in vivo. Mice (C57BL/6J, n = 30) were fed Zn marginal diets (ZM, 5 mg/kg) for 4 weeks, followed by fasting (F) and/or refeeding with ZM or Zn supplemental (300 mg/kg, ZS) diets for 3 or 6 h. Plasma insulin was greater (P < 0.05) in refed animals as compared to F animals. Protein phosphorylation was detected using multiplex analysis and Western blotting. Multiplex analysis indicated greater (P < 0.05) p70 S6 kinase (p70^S6K) and glycogen synthase kinase 3 (GSK-3 α/β) phosphorylation in livers from 6-h refed ZS animals as compared to F animals. Western blots indicated increased (P < 0.05) Akt (Ser 473) phosphorylation in skeletal muscle from animals refed ZS diets for 3 and 6 h as compared to F animals. The ZS diet affected phosphorylation of GSK-3 (α/β) in liver, as 3-h ZS refed animals had greater (P < 0.01) phosphorylation than F animals. These findings indicate that Zn may contribute to the initiation of protein synthesis as a signaling molecule in vivo. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Army or the Department of Defense. Any citations of commercial organizations and trade names in this report do not constitute an official Department of the Army endorsement of approval of the products or services of these organizations.     

Rhodopsin-regulated Insulin Receptor Signaling Pathway in Rod Photoreceptor Neurons

The retina is an integral part of the central nervous system and retinal cells are known to express insulin receptors (IR), although their function is not known. This article describes recent studies that link the photoactivation of rhodopsin to tyrosine phosphorylation of the IR and subsequent activation of phosphoinositide 3-kinase, a neuron survival factor. Our studies suggest that the physiological role of this process is to provide neuroprotection of the retina against light damage by activating proteins that protect against stress-induced apoptosis. We focus mainly on our recently identified regulation of the IR pathway through the G-protein-coupled receptor rhodopsin. Various mutant and knockout proteins of phototransduction cascade have been used to study the light-induced activation of the retinal IR. Our studies suggest that rhodopsin may have additional previously uncharacterized signaling functions in photoreceptors.     

Effects of Fasting and Refeeding on Expression of Atrogin-1 and Akt/FOXO Signaling Pathway in Skeletal Muscle of Chicks

This experiment was conducted to study the effects of fasting and refeeding on expression of the atrogin-1 and Akt/FOXO signaling pathway in skeletal muscle of chicks. Chicks were fasted for 24 h and refed for 2 h. Atrogin-1 mRNA expression was increased by fasting, and their increment was reduced by refeeding. Phosphorylations of Akt and FOXO1 were not decreased by fasting, but, they were increased by refeeding. These results indicate that refeeding stimulates phosphorylation of Akt/FOXO, resulting in a decrease in atrogin-1 expression in skeletal muscle of chicks.     

Characterising the Inhibitory Actions of Ceramide upon Insulin Signaling in Different Skeletal Muscle Cell Models: A Mechanistic Insight

Ceramides are known to promote insulin resistance in a number of metabolically important tissues including skeletal muscle, the predominant site of insulin-stimulated glucose disposal. Depending on cell type, these lipid intermediates have been shown to inhibit protein kinase B (PKB/Akt), a key mediator of the metabolic actions of insulin, via two distinct pathways: one involving the action of atypical protein kinase C (aPKC) isoforms, and the second dependent on protein phosphatase-2A (PP2A). The main aim of this study was to explore the mechanisms by which ceramide inhibits PKB/Akt in three different skeletal muscle-derived cell culture models; rat L6 myotubes, mouse C2C12 myotubes and primary human skeletal muscle cells. Our findings indicate that the mechanism by which ceramide acts to repress PKB/Akt is related to the myocellular abundance of caveolin-enriched domains (CEM) present at the plasma membrane. Here, we show that ceramide-enriched-CEMs are markedly more abundant in L6 myotubes compared to C2C12 myotubes, consistent with their previously reported role in coordinating aPKC-directed repression of PKB/Akt in L6 muscle cells. In contrast, a PP2A-dependent pathway predominantly mediates ceramide-induced inhibition of PKB/Akt in C2C12 myotubes. In addition, we demonstrate for the first time that ceramide engages an aPKC-dependent pathway to suppress insulin-induced PKB/Akt activation in palmitate-treated cultured human muscle cells as well as in muscle cells from diabetic patients. Collectively, this work identifies key mechanistic differences, which may be linked to variations in plasma membrane composition, underlying the insulin-desensitising effects of ceramide in different skeletal muscle cell models that are extensively used in signal transduction and metabolic studies.     

Insulin-Regulated Adenylyl Cyclase Signaling System in Rat Skeletal Muscles under Conditions of in vivo Insulin Administration and of Insulin Insufficiency Produced by Streptozotocin Diabetes

Possibility of the appearance of functional defects in the adenylyl cyclase (AC) signaling mechanism (ACSM) of insulin action, which was discovered by the authors earlier [1–3], is studied in skeletal muscles of rats with acute insulin insufficiency produced by streptozotocin diabetes (24 h). This ACSM includes the signaling chain: receptor-tyrosine kinase Gi-protein phosphatidylinositol 3-kinase protein kinase C-zeta Gs-protein adenylyl cyclase protein kinase A. At comparative evaluation of the functional state of individual molecular blocks of ACSM and the entire mechanism as a whole in skeletal muscles of diabetic rats in comparison with control animals, the following facts have been revealed: (1) an increase of the AC basal activity and a decrease of effects of non-hormonal activators of AC (guanine nucleotides, NaF, forskolin) ; (2) reduction of reactivity of the whole ACSM to insulin (10_–8 M, in vitro) and to combined action of the hormone and GIDP (10_–6 M) ; (3) a decrease of the activating action of insulin on key enzymes of carbohydrate metabolism—glycogen synthase and glucose-6-phosphate dehydrogenase (G6PDG). It is concluded that insulin insufficiency leads to several disturbances in the insulin ACSM: at the level of its catalytic component—AC, Gs protein and its coupling with AC, as well as to a decrease of regulatory metabolic effects of the hormone. These data indicate a decrease of sensitivity of skeletal muscles of diabetic rats to insulin and an involvement of this hormone in maintenance of functionally active status of the ACSM of insulin signal transduction.     

Muscle Atrophy in Response to Cytotoxic Chemotherapy Is Dependent on Intact Glucocorticoid Signaling in Skeletal Muscle

Cancer cachexia is a syndrome of weight loss that results from the selective depletion of skeletal muscle mass and contributes significantly to cancer morbidity and mortality. The driver of skeletal muscle atrophy in cancer cachexia is systemic inflammation arising from both the cancer and cancer treatment. While the importance of tumor derived inflammation is well described, the mechanism by which cytotoxic chemotherapy contributes to cancer cachexia is relatively unexplored. We found that the administration of chemotherapy to mice produces a rapid inflammatory response. This drives activation of the hypothalamic-pituitary-adrenal axis, which increases the circulating level of corticosterone, the predominant endogenous glucocorticoid in rodents. Additionally, chemotherapy administration results in a significant loss of skeletal muscle mass 18 hours after administration with a concurrent induction of genes involved with the ubiquitin proteasome and autophagy lysosome systems. However, in mice lacking glucocorticoid receptor expression in skeletal muscle, chemotherapy-induced muscle atrophy is completely blocked. This demonstrates that cytotoxic chemotherapy elicits significant muscle atrophy driven by the production of endogenous glucocorticoids. Further, it argues that pharmacotherapy targeting the glucocorticoid receptor, given in concert with chemotherapy, is a viable therapeutic strategy in the treatment of cancer cachexia.     

Regulation of mTOR by Mechanically Induced Signaling Events in Skeletal Muscle

Mechanical stimuli play a major role in the regulation of skeletal muscle mass, and themaintenance of muscle mass contributes significantly to disease prevention and the quality oflife. Although a link between mechanical stimuli and the regulation of muscle mass has beenrecognized for decades, the mechanisms involved in converting mechanical information into themolecular events that control this process have not been defined. Nevertheless, significantadvancements are being made in this field, and it has recently been established that signalingthrough a rapamycin-sensitive pathway is necessary for mechanically induced growth of skeletalmuscle. Since rapamycin is a highly specific inhibitor of a protein kinase called the mammaliantarget of rapamycin (mTOR), many investigators have concluded that mTOR signaling isnecessary for the mechanically induced growth of skeletal muscle. In this review, we havesummarized the current knowledge regarding how mechanical stimuli activate mTOR signaling,discussed the newly discovered role of phospholipase D (PLD) and phosphatidic acid (PA) inthis pathway, and considered the potential roles of PLD and PA in the mechanical regulation ofskeletal muscle mass.     

Sustained Action of Ceramide on the Insulin Signaling Pathway in Muscle Cells: IMPLICATION OF THE DOUBLE-STRANDED RNA-ACTIVATED PROTEIN KINASE*

In vivo, ectopic accumulation of fatty acids in muscles leads to alterations in insulin signaling at both the IRS1 and Akt steps. However, in vitro treatments with saturated fatty acids or their derivative ceramide demonstrate an effect only at the Akt step. In this study, we adapted our experimental procedures to mimic the in vivo situation and show that the double-stranded RNA-dependent protein kinase (PKR) is involved in the long-term effects of saturated fatty acids on IRS1. C2C12 or human muscle cells were incubated with palmitate or directly with ceramide for short or long periods, and insulin signaling pathway activity was evaluated. PKR involvement was assessed through pharmacological and genetic studies. Short-term treatments of myotubes with palmitate, a ceramide precursor, or directly with ceramide induce an inhibition of Akt, whereas prolonged periods of treatment show an additive inhibition of insulin signaling through increased IRS1 serine 307 phosphorylation. PKR mRNA, protein, and phosphorylation are increased in insulin-resistant muscles. When PKR activity is reduced (siRNA or a pharmacological inhibitor), serine phosphorylation of IRS1 is reduced, and insulin-induced phosphorylation of Akt is improved. Finally, we show that JNK mediates ceramide-activated PKR inhibitory action on IRS1. Together, in the long term, our results show that ceramide acts at two distinct levels of the insulin signaling pathway (IRS1 and Akt). PKR, which is induced by both inflammation signals and ceramide, could play a major role in the development of insulin resistance in muscle cells.     

原代培养骨骼肌细胞胰岛素抵抗模型的建立

目的:观察高脂负荷在胰岛素抵抗形成中的意义.方法:用不同浓度椋榈酸、胰岛素分别培养骨骼肌细胞2h、6h、12h、24h,对棕榈酸诱导组的一部分细胞给予1× 10-7M胰岛素刺激2h,用血糖检测试剂盒(GOD-POD)检测各组培养液中的葡萄糖含量,研究不同浓度棕榈酸、胰岛素对骨骼肌细胞摄取葡萄糖的影响,观察胰岛素的生理功效的变化.结果:0.6mM棕榈酸诱导12h以上或者5×101-M胰岛素诱导24h后,培养液中的葡萄糖浓度比正常组高且有显著性差异,表明细胞的糖代谢能力降低.经1×10-7M胰岛素刺激2h后的棕榈酸诱导组,培养液中葡萄糖的浓度与未经胰岛素刺激的棕榈酸诱导组相比无显著差异,胰岛素的生理功效降低,证实棕榈酸诱导组细胞已对胰岛素产生耐受.结论:高脂或高胰岛素条件均可诱导原代骨骼肌细胞胰岛素抵抗模型.     

Lipid-induced Muscle Insulin Resistance Is Mediated by GGPPS via Modulation of the RhoA/Rho Kinase Signaling Pathway

Elevated circulating free fatty acid levels are important contributors to insulin resistance in the muscle and liver, but the underlying mechanisms require further elucidation. Here, we show that geranylgeranyl diphosphate synthase 1 (GGPPS), which is a branch point enzyme in the mevalonic acid pathway, promotes lipid-induced muscle insulin resistance through activation of the RhoA/Rho kinase signaling pathway. We have found that metabolic perturbation would increase GGPPS expression in the skeletal muscles of db/db mice and high fat diet-fed mice. To address the metabolic effects of GGPPS activity in skeletal muscle, we generated mice with specific GGPPS deletions in their skeletal muscle tissue. Heterozygous knock-out of GGPPS in the skeletal muscle improved systemic insulin sensitivity and glucose homeostasis in mice fed both normal chow and high fat diets. These metabolic alterations were accompanied by activated PI3K/Akt signaling and enhanced glucose uptake in the skeletal muscle. Further investigation showed that the free fatty acid-stimulated GGPPS expression in the skeletal muscle was able to enhance the geranylgeranylation of RhoA, which further induced the inhibitory phosphorylation of IRS-1 (Ser-307) by increasing Rho kinase activity. These results implicate a crucial role of the GGPPS/RhoA/Rho kinase/IRS-1 pathway in skeletal muscle, in which it mediates lipid-induced systemic insulin resistance in obese mice. Therefore, skeletal muscle GGPPS may represent a potential pharmacological target for the prevention and treatment of obesity-related type 2 diabetes.