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.     

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.     

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.     

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.     

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.     

Effect of Sfrp5 on Cytokine Release and Insulin Action in Primary Human Adipocytes and Skeletal Muscle Cells

Secreted frizzled-related protein 5 (Sfrp5) is an adipokine with anti-inflammatory and insulin-sensitizing properties in mice. However, the mechanism of Sfrp5 action, especially in humans, is largely unknown. Therefore, cytokine release and insulin signaling were analyzed to investigate the impact of Sfrp5 on inflammation and insulin signaling in primary human adipocytes and skeletal muscle cells (hSkMC). Sfrp5 neither affected interleukin (IL)-6, monocyte chemoattractant protein-1 (MCP-1) and adiponectin release from human adipocytes, nor IL-6 and IL-8 release from hSkMC. In tumor necrosis factor (TNF) α-treated adipocytes, Sfrp5 reduced IL-6 release by 49% (p<0.05), but did not affect MCP-1 and adiponectin release. In MCP-1-treated hSkMC, Sfrp5 did not affect cytokine secretion. In untreated adipocytes, Sfrp5 decreased the insulin-mediated phosphorylation of Akt-Ser473, Akt-Thr308, GSK3α-Ser21 and PRAS40-Thr246 by 34% (p<0.01), 31% (p<0.05), 37% (p<0.05) and 34% (p<0.01), respectively, and the stimulation of glucose uptake by 25% (p<0.05). Incubation with TNFα increased the phosphorylation of JNK and NFκB, and impaired insulin signaling. When Sfrp5 and TNFα were combined, there was no additional effect on insulin signaling and JNK phosphorylation, but phosphorylation of NFκB was reversed to basal levels. Sfrp5 had no effect on insulin signaling in untreated or in MCP-1 treated hSkMC. Thus, Sfrp5 lowered IL-6 release and NFκB phosphorylation in cytokine-treated human adipocytes, but not under normal conditions, and decreased insulin signaling in untreated human adipocytes. Sfrp5 did not act on hSkMC. Therefore, the cellular actions of Sfrp5 seem to depend on the type of tissue as well as its inflammatory and metabolic state.     

胆固醇逆转运相关蛋白基因在骨骼肌的表达

构建载脂蛋白ＡＩ（ａｐｏＡＩ）、载脂蛋白Ｅ（ａｐｏＥ）和卵磷脂胆固醇酰基转移酶（ＬＣＡＴ）的病毒或非病毒载体,分别转染离体成肌细胞或直接注入小鼠骨骼肌,观察其表达程序及分泌人血等情况,探讨借用骨骼肌异源表达这些在胆固醇逆转运中起关键作用的重要候选基因,进而发展一种简易安全基因治疗方法的可能性。结果显示,质粒表达载体ｐＣＭＶａｐｏＥ３和重组腺病毒载体Ａｄ－ＲＳＶ－ａｐｏＡＩ在原代培养小鼠成肌细胞和成     

Angiotensin Type 2 Receptor Signaling in Satellite Cells Potentiates Skeletal Muscle Regeneration

Patients with advanced congestive heart failure (CHF) or chronic kidney disease (CKD) often have increased angiotensin II (Ang II) levels and cachexia. Ang II infusion in rodents causes sustained skeletal muscle wasting and decreases muscle regenerative potential through Ang II type 1 receptor (AT1R)-mediated signaling, likely contributing to the development of cachexia in CHF and CKD. However, the potential role of Ang II type 2 receptor (AT2R) signaling in skeletal muscle physiology is unknown. We found that AT2R expression was increased robustly in regenerating skeletal muscle after cardiotoxin (CTX)-induced muscle injury in vivo and differentiating myoblasts in vitro, suggesting that the increase in AT2R played an important role in regulating myoblast differentiation and muscle regeneration. To determine the potential role of AT2R in muscle regeneration, we infused C57BL/6 mice with the AT2R antagonist PD123319 during CTX-induced muscle regeneration. PD123319 reduced the size of regenerating myofibers and expression of the myoblast differentiation markers myogenin and embryonic myosin heavy chain. On the other hand, AT2R agonist {"type":"entrez-protein","attrs":{"text":"CGP42112","term_id":"874777115","term_text":"CGP42112"}}CGP42112 infusion potentiated CTX injury-induced myogenin and embryonic myosin heavy chain expression and increased the size of regenerating myofibers. In cultured myoblasts, AT2R knockdown by siRNA suppressed myoblast differentiation marker expression and myoblast differentiation via up-regulation of phospho-ERK1/2, and ERK inhibitor treatment completely blocked the effect of AT2R knockdown. These data indicate that AT2R signaling positively regulates myoblast differentiation and potentiates skeletal muscle regenerative potential, providing a new therapeutic target in wasting disorders such as CHF and CKD.     

棕榈酸的组织吸收分布及对骨骼肌胰岛素抵抗的影响

脂肪酸代谢紊乱是Ⅱ型糖尿病的主要致病因素之一。棕榈酸是血液中含量最高的游离脂肪酸。我们建立了大鼠颈静脉置管输注棕榈酸的模型,发现血液中的大部分棕榈酸被骨骼肌组织所吸收。以棕榈酸处理的C2C12骨骼肌细胞为实验模型发现,棕榈酸进入骨骼肌细胞后的中间代谢产物(磷脂和甘油二酯)的累积,会造成内质网应激及胰岛素抵抗。提示血液中棕榈酸含量的升高可能通过骨骼肌的胰岛素抵抗机制,影响Ⅱ型糖尿病的发生和发展。     

Visualization of Ca2+ Signaling During Embryonic Skeletal Muscle Formation in Vertebrates

Dynamic changes in cytosolic and nuclear Ca²⁺ concentration are reported to play a critical regulatory role in different aspects of skeletal muscle development and differentiation. Here we review our current knowledge of the spatial dynamics of Ca²⁺ signals generated during muscle development in mouse, rat, and Xenopus myocytes in culture, in the exposed myotome of dissected Xenopus embryos, and in intact normally developing zebrafish. It is becoming clear that subcellular domains, either membrane-bound or otherwise, may have their own Ca²⁺ signaling signatures. Thus, to understand the roles played by myogenic Ca²⁺ signaling, we must consider: (1) the triggers and targets within these signaling domains; (2) interdomain signaling, and (3) how these Ca²⁺ signals integrate with other signaling networks involved in myogenesis. Imaging techniques that are currently available to provide direct visualization of these Ca²⁺ signals are also described.The recognition of Ca²⁺ as a key regulator of muscle contraction dates back to Sydney Ringer''s seminal observations in the latter part of the 19th Century (Ringer 1883; Ringer 1886; Ringer and Buxton 1887; see reviews by Martonosi 2000; Szent-Györgyi 2004). More recently, evidence is steadily accumulating to support the proposition that Ca²⁺ also plays a necessary and essential role in regulating embryonic muscle development and differentiation (Flucher and Andrews 1993; Ferrari et al. 1996; Lorenzon et al. 1997; Ferrari and Spitzer 1998, 1999; Wu et al. 2000; Powell et al. 2001; Jaimovich and Carrasco 2002; Li et al. 2004; Brennan et al. 2005; Harris et al. 2005; Campbell et al. 2006; Terry et al. 2006; Fujita et al. 2007; and see reviews by Berchtold et al. 2000; Ferrari et al. 2006; Al-Shanti and Stewart 2009). What is currently lacking, however, is extensive direct visualization of the spatial dynamics of the Ca²⁺ signals generated by developing and differentiating muscle cells. This is especially so concerning in situ studies. The object of this article, therefore, is to review and report the current state of our understanding concerning the spatial nature of Ca²⁺ signaling during embryonic muscle development, especially from an in vivo perspective, and to suggest possible directions for future research. The focus of our article is embryonic skeletal muscle development because of this being an area of significant current interest. Several of the basic observations reported, however, may also be common to cardiac muscle development and in some cases to smooth muscle development. What the recent development of reliable imaging techniques has most certainly done, is to add an extra dimension of complexity to understanding the roles played by Ca²⁺ signaling in skeletal muscle development. For example, it is clear that membrane-bound subcellular compartments, such as the nucleus (Jaimovich and Carrasco 2002), may have endogenous Ca²⁺ signaling activities, as do specific cytoplasmic domains, such as the subsarcolemmal space (Campbell et al. 2006). How these Ca²⁺ signals interact with specific down-stream targets within their particular domain, and how they might serve to communicate information among domains, will most certainly be one of the future challenges in elucidating the Ca²⁺-mediated regulation of muscle development.Any methodology used to study the properties of biological molecules and how they interact during development should ideally provide spatial information, because researchers increasingly need to integrate data about the interactions that underlie a biological process (such as differentiation) with information regarding the precise location within cells or an embryo where these interactions take place. Current Ca²⁺ imaging techniques are beginning to provide us with this spatial information, and are thus opening up exciting new avenues of investigation in our quest to understand the signaling pathways that regulate muscle development (

The Rho-Guanine Nucleotide Exchange Factor Domain of Obscurin Activates RhoA Signaling in Skeletal Muscle

Obscurin is a large (∼800-kDa), modular protein of striated muscle that concentrates around the M-bands and Z-disks of each sarcomere, where it is well positioned to sense contractile activity. Obscurin contains several signaling domains, including a rho-guanine nucleotide exchange factor (rhoGEF) domain and tandem pleckstrin homology domain, consistent with a role in rho signaling in muscle. We investigated the ability of obscurin''s rhoGEF domain to interact with and activate small GTPases. Using a combination of in vitro and in vivo approaches, we found that the rhoGEF domain of obscurin binds selectively to rhoA, and that rhoA colocalizes with obscurin at the M-band in skeletal muscle. Other small GTPases, including rac1 and cdc42, neither associate with the rhoGEF domain of obscurin nor concentrate at the level of the M-bands. Furthermore, overexpression of the rhoGEF domain of obscurin in adult skeletal muscle selectively increases rhoA expression and activity in this tissue. Overexpression of obscurin''s rhoGEF domain and its effects on rhoA alter the expression of rho kinase and citron kinase, both of which can be activated by rhoA in other tissues. Injuries to rodent hindlimb muscles caused by large-strain lengthening contractions increases rhoA activity and displaces it from the M-bands to Z-disks, similar to the effects of overexpression of obscurin''s rhoGEF domain. Our results suggest that obscurin''s rhoGEF domain signals at least in part by inducing rhoA expression and activation, and altering the expression of downstream kinases in vitro and in vivo.