Denervation atrophy is independent from Akt and mTOR activation and is not rescued by myostatin inhibition

The purpose of our study was to compare two acquired muscle atrophies and the use of myostatin inhibition for their treatment. Myostatin naturally inhibits skeletal muscle growth by binding to ActRIIB, a receptor on the cell surface of myofibers. Because blocking myostatin in an adult wild-type mouse induces profound muscle hypertrophy, we applied a soluble ActRIIB receptor to models of disuse (limb immobilization) and denervation (sciatic nerve resection) atrophy. We found that treatment of immobilized mice with ActRIIB prevented the loss of muscle mass observed in placebo-treated mice. Our results suggest that this protection from disuse atrophy is regulated by serum and glucocorticoid-induced kinase (SGK) rather than by Akt. Denervation atrophy, however, was not protected by ActRIIB treatment, yet resulted in an upregulation of the pro-growth factors Akt, SGK and components of the mTOR pathway. We then treated the denervated mice with the mTOR inhibitor rapamycin and found that, despite a reduction in mTOR activation, there is no alteration of the atrophy phenotype. Additionally, rapamycin prevented the denervation-induced upregulation of the mTORC2 substrates Akt and SGK. Thus, our studies show that denervation atrophy is not only independent from Akt, SGK and mTOR activation but also has a different underlying pathophysiological mechanism than disuse atrophy.KEY WORDS: Skeletal muscle, Muscle atrophy pathophysiology, TGF-β signaling, Myostatin, Denervation atrophy     

Chronic paraplegia-induced muscle atrophy downregulates the mTOR/S6K1 signaling pathway.

Ribosomal S6 kinase 1 (S6K1) is a downstream component of the mammalian target of rapamycin (mTOR) signaling pathway and plays a regulatory role in translation initiation, protein synthesis, and muscle hypertrophy. AMP-activated protein kinase (AMPK) is a cellular energy sensor, a negative regulator of mTOR, and an inhibitor of protein synthesis. The purpose of this study was to determine whether the hypertrophy/cell growth-associated mTOR pathway was downregulated during muscle atrophy associated with chronic paraplegia. Soleus muscle was collected from male Sprague-Dawley rats 10 wk following complete T(4)-T(5) spinal cord transection (paraplegic) and from sham-operated (control) rats. We utilized immunoprecipitation and Western blotting techniques to measure upstream [AMPK, Akt/protein kinase B (PKB)] and downstream components of the mTOR signaling pathway [mTOR, S6K1, SKAR, 4E-binding protein 1 (4E-BP1), and eukaryotic initiation factor (eIF) 4G and 2alpha]. Paraplegia was associated with significant soleus muscle atrophy (174 +/- 8 vs. 240 +/- 13 mg; P < 0.05). There was a reduction in phosphorylation of mTOR, S6K1, and eIF4G (P < 0.05) with no change in Akt/PKB or 4E-BP1 (P > 0.05). Total protein abundance of mTOR, S6K1, eIF2alpha, and Akt/PKB was decreased, and increased for SKAR (P < 0.05), whereas 4E-BP1 and eIF4G did not change (P > 0.05). S6K1 activity was significantly reduced in the paraplegic group (P < 0.05); however, AMPKalpha2 activity was not altered (3.5 +/- 0.4 vs. 3.7 +/- 0.5 pmol x mg(-1) x min(-1), control vs. paraplegic rats). We conclude that paraplegia-induced muscle atrophy in rats is associated with a general downregulation of the mTOR signaling pathway. Therefore, in addition to upregulation of atrophy signaling during muscle wasting, downregulation of muscle cell growth/hypertrophy-associated signaling appears to be an important component of long-term muscle loss.     

缺氧诱导因子1与PI3K/Akt/mTOR信号转导通路

缺氧诱导因子1（HIF-1）是参与缺氧调节的核心因子,可调控一系列缺氧诱导基因的表达,与机体许多生理和病理过程也密切相关。尽管一些研究显示缺氧和非缺氧性刺激可通过PI3K/Akt/mTOR信号途径诱导HIF-1的表达和活性,PI3K信号途径是否参与对HIF-1的调节仍然是个有争议的研究热点。明确HIF-1和PI3K的相互作用关系,能进一步为肿瘤等相关疾病的防治提供新的思路和方法。本文主要就HIF-1和PI3K/Akt/mTOR关系作一简要综述。     

Altered calcium signaling in colonic smooth muscle of type 1 diabetic mice

Seventy-six percent of diabetic patients develop gastrointestinal symptoms, such as constipation. However, the direct effects of diabetes on intestinal smooth muscle are poorly described. This study aimed to identify the role played by smooth muscle in mediating diabetes-induced colonic dysmotility. To induce type 1 diabetes, mice were injected intraperitoneally with low-dose streptozotocin once a day for 5 days. Animals developed hyperglycemia (>200 mg/dl) 1 wk after the last injection and were euthanized 7-8 wk after the last treatment. Computed tomography demonstrated decreased overall gastrointestinal motility in the diabetic mice. In vitro contractility of colonic smooth muscle rings from diabetic mice was also decreased. Fura-2 ratiometric Ca(2+) imaging showed attenuated Ca(2+) increases in response to KCl stimulation that were associated with decreased light chain phosphorylation in diabetic mice. The diabetic mice also exhibited elevated basal Ca(2+) levels, increased myosin phosphatase targeting subunit 1 expression, and significant changes in expression of Ca(2+) handling proteins, as determined by quantitative RT-PCR and Western blotting. Mice that were hyperglycemic for <1 wk also showed decreased colonic contractile responses that were associated with decreased Ca(2+) increases in response to KCl stimulation, although without an elevation in basal Ca(2+) levels or a significant change in the expression of Ca(2+) signaling molecules. These data demonstrate that type 1 diabetes is associated with decreased depolarization-induced Ca(2+) influx in colonic smooth muscle that leads to attenuated myosin light chain phosphorylation and impaired colonic contractility.     

PI3K/Akt/mTOR signaling regulates glutamate transporter 1 in astrocytes

Reduction in or dysfunction of glutamate transporter 1 (GLT1) is linked to several neuronal disorders such as stroke, Alzheimer’s disease, and amyotrophic lateral sclerosis. However, the detailed mechanism underlying GLT1 regulation has not been fully elucidated. In the present study, we first demonstrated the effects of mammalian target of rapamycin (mTOR) signaling on GLT1 regulation. We prepared astrocytes cultured in astrocyte-defined medium (ADM), which contains several growth factors including epidermal growth factor (EGF) and insulin. The levels of phosphorylated Akt (Ser473) and mTOR (Ser2448) increased, and GLT1 levels were increased in ADM-cultured astrocytes. Treatment with a phosphatidylinositol 3-kinase (PI3K) inhibitor or an Akt inhibitor suppressed the phosphorylation of Akt (Ser473) and mTOR (Ser2448) as well as decreased ADM-induced GLT1 upregulation. Treatment with the mTOR inhibitor rapamycin decreased GLT1 protein and mRNA levels. In contrast, rapamycin did not affect Akt (Ser473) phosphorylation. Our results suggest that mTOR is a downstream target of the PI3K/Akt pathway regulating GLT1 expression.     

Ethanolic Ginkgo biloba leaf extract prevents renal fibrosis through Akt/mTOR signaling in diabetic nephropathy

BackgroundRecently, extract of Ginkgo biloba leaves (GbE) have become widely known phytomedicines and have shown various pharmacological activities, including improvement of blood circulation, protection of oxidative cell damage, prevention of Alzheimer's disease, treatment of cardiovascular disease and diabetes complications. This study was designed to investigate the effects of an ethanolic GbE on renal fibrosis in diabetic nephropathy (DN) and to clarify the possible mechanism by which GbE prevents renal fibrosis.Study designWe investigated the protective effects of GbE on renal fibrosis in STZ-induced diabetic rats. Rats were randomized into six groups termed normal control, diabetes mellitus, low dose of GbE (50 mg/kg/d), intermediate dose of GbE (100 mg/kg/d), high dose of GbE (200 mg/kg/d) and rapamycin (1 mg/kg/d).MethodsAfter 12 weeks, the rats were sacrificed and then fasting blood glucose (FBG), creatinine (Cr), blood urea nitrogen (BUN), urine protein, relative kidney weight, glycogen and collagen accumulation, and collagen IV and laminin expression were measured by different methods. The amounts of E-cadherin, α-SMA and snail, as well as the phosphorylation of Akt, mTOR and p70S6K in the renal cortex of rats, were examined by western blotting.ResultsCompared with diabetic rats, the levels of Cr, BUN, urine protein, relative kidney weight, accumulation of glycogen and collagen, and expression of collagen IV and laminin in the renal cortex were all decreased in GbE treated rats. In addition, GbE reduced the expression of E-cadherin, α-SMA, snail and the phosphorylation of Akt, mTOR and p70S6K in diabetic renal cortex.ConclusionGbE can prevent renal fibrosis in rats with diabetic nephropathy, which is most likely to be associated with its abilities to inhibit the Akt/mTOR signaling pathway.     

MiR-28 inhibits cardiomyocyte survival through suppressing PDK1/Akt/mTOR signaling

MicroRNAs play critical roles in regulating cell survival under multiple pathological conditions of heart diseases. Oxidative stress-induced apoptosis contributes greatly to heart ischemia-reperfusion injury. Herein, we describe a novel regulatory role of miR-28 on the survival of cardiomyocytes. We show that miR-28 was upregulated in cardiomyocytes treated with hydrogen peroxide (H₂O₂). MiR-28 gain of function sensitized cell apoptosis, whereas miR-28 loss of function partially rescued cell apoptosis induced by H₂O₂. Importantly, we observed a significant reduction in Akt/mammalian target of rapamycin (mTOR) signaling activity after miR-28 treatment. Luciferase activity assay and western blot analysis both revealed that, phosphoinositide-dependent kinase-1 (PDK1), which is critical for Akt activation, was directly and negatively modulated by miR-28. Our results therefore indicate that miR-28 regulates oxidative stress-induced cell apoptosis in heart muscle cells, which possibly involves a PDK1/Akt/mTOR-dependent mechanism. MIR-28 could serve as a critical therapeutic target to diminish oxidative stress-induced cell death in the heart.     

Nrf2 modulates contractile and metabolic properties of skeletal muscle in streptozotocin-induced diabetic atrophy

The role of Nrf2 in disease prevention and treatment is well documented; however the specific role of Nrf2 in skeletal muscle is not well described. The current study investigated whether Nrf2 plays a protective role in an STZ-induced model of skeletal muscle atrophy.     

Deciphering the signaling pathways of cancer stem cells of glioblastoma multiforme: role of Akt/mTOR and MAPK pathways

These findings emphasize that the mTOR pathway may contribute to maintenance of quiescence of CSCs, and provide a basis for manipulating CSCs in the treatment of GBM. Future research should focus on further defining the PI3K/Akt/mTOR molecular network in the regulation of stem cell quiescence and provide rationale for targeting the cancer-initiating cells of GBM.     

Down‐regulation of the mitochondrial i‐AAA protease Yme1L induces muscle atrophy via FoxO3a and myostatin activation

Muscle atrophy is closely associated with many diseases, including diabetes and cardiac failure. Growing evidence has shown that mitochondrial dysfunction is related to muscle atrophy; however, the underlying mechanisms are still unclear. To elucidate how mitochondrial dysfunction causes muscle atrophy, we used hindlimb‐immobilized mice. Mitochondrial function is optimized by balancing mitochondrial dynamics, and we observed that this balance shifted towards mitochondrial fission and that MuRF1 and atrogin‐1 expression levels were elevated in these mice. We also found that the expression of yeast mitochondrial escape 1‐like ATPase (Yme1L), a mitochondrial AAA protease was significantly reduced both in hindlimb‐immobilized mice and carbonyl cyanide m‐chlorophenylhydrazone (CCCP)‐treated C2C12 myotubes. When Yme1L was depleted in myotubes, the short form of optic atrophy 1 (Opa1) accumulated, leading to mitochondrial fragmentation. Moreover, a loss of Yme1L, but not of LonP1, activated AMPK and FoxO3a and concomitantly increased MuRF1 in C2C12 myotubes. Intriguingly, the expression of myostatin, a myokine responsible for muscle protein degradation, was significantly increased by the transient knock‐down of Yme1L. Taken together, our results suggest that a deficiency in Yme1L and the consequential imbalance in mitochondrial dynamics result in the activation of FoxO3a and myostatin, which contribute to the pathological state of muscle atrophy.     

Formononetin ameliorates muscle atrophy by regulating myostatin-mediated PI3K/Akt/FoxO3a pathway and satellite cell function in chronic kidney disease

Muscle atrophy is a common complication in chronic kidney disease (CKD). Inflammation and myostatin play important roles in CKD muscle atrophy. Formononetin (FMN), which is a major bioactive isoflavone compound in Astragalus membranaceus, exerts anti-inflammatory effects and the promotion of myogenic differentiation. Our study is based on myostatin to explore the effects and mechanisms of FMN in relation to CKD muscle atrophy. In this study, CKD rats and tumour necrosis factor α (TNF-α)-induced C2C12 myotubes were used for in vivo and in vitro models of muscle atrophy. The results showed that FMN significantly improved the renal function, nutritional status and inflammatory markers in CKD rats. Values for bodyweight, weight of tibialis anterior and gastrocnemius muscles, and cross-sectional area (CSA) of skeletal muscles were significantly larger in the FMN treatment rats. Furthermore, FMN significantly suppressed the expressions of MuRF-1, MAFbx and myostatin in the muscles of CKD rats and the TNF-α-induced C2C12 myotubes. Importantly, FMN significantly increased the phosphorylation of PI3K, Akt, and FoxO3a and the expressions of the myogenic proliferation and differentiation markers, myogenic differentiation factor D (MyoD) and myogenin in muscles of CKD rats and the C2C12 myotubes. Similar results were observed in TNF-α-induced C2C12 myotubes transfected with myostatin-small interfering RNA (si-myostatin). Notably, myostatin overexpression plasmid (myostatin OE) abolished the effect of FMN on the phosphorylation of the PI3K/Akt/FoxO3a pathway and the expressions of MyoD and myogenin. Our findings suggest that FMN ameliorates muscle atrophy related to myostatin-mediated PI3K/Akt/FoxO3a pathway and satellite cell function.     

Myostatin信号通路在4周离心耐力运动改善2型糖尿病大鼠骨骼肌萎缩中的作用

目的：观察4周离心耐力运动对2型糖尿病大鼠代谢障碍及肌萎缩的影响,探讨myostatin/Smad3/atrogin-1信号通路在肌萎缩中的作用。方法：9周高脂饲养联合STZ注射建立2型糖尿病大鼠模型。将普通饲料组大鼠随机分为对照组（C,n=6）和运动组（E,n=9;将2型糖尿病模型组大鼠随机分为糖尿病对照组（D,n=8）和糖尿病运动组（DE,n=12）。运动方案：坡度-5°,跑速16 m/min,每次60 min、每日一次,每周训练5 d,连续4周。最后一次运动后禁食12 h,测定空腹血糖（FBG）、空腹胰岛素（FINS）,计算稳态模式胰岛素抵抗指数（HOMA-IR）和胰岛素敏感指数（ISI）,进行葡萄糖耐量试验。取比目鱼肌观察肌萎缩现象并检测myostatin、Smad3、p-Smad3和atrogin-1表达情况。结果：①与对照组相比,糖尿病组大鼠体重、比目鱼肌质量/胫骨长和肌纤维平均横截面积、FINS和ISI显著降低（P<0.01）,FBG、HOMA-IR和血糖曲线下面积（AUC_BG）以及myostatin、Smad3、p-Smad3、atrogin-1表达均显著升高（P<0.01）。②4周离心运动后,与糖尿病组相比,糖尿病运动组大鼠肌纤维平均横截面积显著升高（P<0.01）,AUC_BG、HOMA-IR及myostatin、p-Smad3、atrogin-1表达显著降低（P<0.05,P<0.01）。结论：myostatin/Smad3/atrogin-1信号通路上调是导致2型糖尿病肌萎缩的重要原因,4周离心耐力运动可能通过下调myostatin、p-Smad3和atrogin-1表达抑制肌萎缩,进而改善2型糖尿病代谢障碍,提高胰岛素敏感性。     

mTOR and Akt signaling in cancer: SGK cycles in

In a recent issue of Molecular Cell, Hong et al. (2008) describe an alternative mechanism by which mTOR promotes cell-cycle progression; it phosphorylates and activates SGK, which in turn phosphorylates the cell-cycle inhibitor p27, promoting its cytoplasmic retention.     

Knockdown of insulin receptor substrate 1 reduces proliferation and downregulates Akt/mTOR and MAPK pathways in K562 cells

BCR-ABL kinase activates downstream signaling pathways, including the PI3K-Akt/mTOR and the MAPK pathway. IRS1 has been previously described as constitutively phosphorylated and associated with BCR-ABL in K562 cells, suggesting that IRS1 has role in the BCR-ABL signaling pathways. In this study, we analyzed the effect of IRS1 silencing, by shRNA-lentiviral delivery, in K562 cells, a CML cell line that presents the BCR-ABL. IRS1 silencing decreased cell proliferation and colony formation in K562 cells, which correlates with the delay of these cells at the G0/G1 phase and a decrease in the S phase of the cell cycle. Furthermore, IRS1 silencing in K562 cells resulted in a decrease of Akt, P70S6K and ERK1/2 phosphorylation. Nevertheless, apoptosis was unaffected by IRS1 knockdown and no alterations were found in the phosphorylation of BAD and in the expression of BCL2 and BAX. BCR-ABL and CRKL phosphorylation levels remained unaffected upon IRS1 silencing, and no synergistic effect was observed with imatinib treatment and IRS1 knockdown, indicating that IRS1 is downstream from BCR-ABL. In conclusion, we demonstrated that inhibition of IRS1 is capable of inducing the downregulation of Akt/mTOR and MAPK pathways and further decreasing proliferation, and clonogenicity and induces to cell cycle delay at G0/G1 phase in BCR-ABL cells.