Insulin receptor substrate-2 proteasomal degradation mediated by a mammalian target of rapamycin (mTOR)-induced negative feedback down-regulates protein kinase B-mediated signaling pathway in beta-cells

Regulation of insulin receptor substrate (IRS)-2 expression is critical to beta-cell survival, but the mechanisms that control this are complex and undefined. Here in pancreatic beta-cells (INS-1), chronic exposure (>8 h) to 15 mm glucose and/or 5 nm IGF-1, increased Ser/Thr phosphorylation of IRS-2, which correlated with decreased IRS-2 levels. This glucose/IGF-1-induced decrease in IRS-2 levels was prevented by the proteasomal inhibitor, lactacystin. In addition, the glucose/IGF-1-induced increase in Ser/Thr phosphorylation of IRS-2 and the subsequent decrease in INS-1 cell IRS-2 protein levels was thwarted by the mammalian target of rapamycin(mTOR) inhibitor, rapamycin. Moreover, adenoviral-mediated expression of constitutively active mTOR (mTORDelta) further increased glucose/IGF-1-induced Ser/Thr phosphorylation of IRS-2 and decreased IRS-2 protein levels, whereas adenoviral-mediated expression of "kinase-dead" mTOR (mTOR-KD) conversely reduced Ser/Thr phosphorylation of IRS-2 and maintained IRS-2 protein levels. In adenoviral-infected beta-cells expressing mTORDelta, the decrease in IRS-2 protein levels was also prevented by rapamycin or lactacystin, further indicating a proteasomal mediated degradation of IRS-2 mediated via mTOR-induced Ser/Thr phosphorylation of IRS-2. Finally, we found that chronic activation of mTOR leading to decreased levels of IRS-2 in INS-1 cells led to a significant decrease in PKB activation and consequently increased beta-cell apoptosis. Thus, chronic activation of mTOR by glucose (and/or IGF-1) in beta-cells leads to increased Ser/Thr phosphorylation of IRS-2 that targets it for proteasomal degradation, resulting in decreased IRS-2 expression and increased beta-cell apoptosis. This may be a contributing mechanism as to how beta-cell mass is decreased by chronic hyperglycemia in the pathogenesis of type-2 diabetes.     

UV-B induced transcript accumulation of DAHP synthase in suspension-cultured Catharanthus roseus cells

Background

AMP-activated protein kinase (AMPK) is a fuel-sensing enzyme that is activated when cells experience energy deficiency and conversely suppressed in surfeit of energy supply. AMPK activation improves insulin sensitivity via multiple mechanisms, among which AMPK suppresses mTOR/S6K-mediated negative feedback regulation of insulin signaling.

Results

In the present study we further investigated the mechanism of AMPK-regulated insulin signaling. Our results showed that 5-aminoimidazole-4-carboxamide-1 ribonucleoside (AICAR) greatly enhanced the ability of insulin to stimulate the insulin receptor substrate-1 (IRS1)-associated PI3K activity in differentiated 3T3-F442a adipocytes, leading to increased Akt phosphorylation at S473, whereas insulin-stimulated activation of mTOR was diminished. In 3T3-F442a preadipocytes, these effects were attenuated by expression of a dominant negative mutant of AMPK α1 subunit. The enhancing effect of ACIAR on Akt phosphorylation was also observed when the cells were treated with EGF, suggesting that it is regulated at a step beyond IR/IRS1. Indeed, when the cells were chronically treated with AICAR in the absence of insulin, Akt phosphorylation was progressively increased. This event was associated with an increase in levels of phosphatidylinositol -3,4,5-trisphosphate (PIP3) and blocked by Wortmannin. We then expressed the dominant negative mutant of PTEN (C124S) and found that the inhibition of endogenous PTEN per se did not affect phosphorylation of Akt at basal levels or upon treatment with AICAR or insulin. Thus, this result suggests that AMPK activation of Akt is not mediated by regulating phosphatase and tensin homologue (PTEN).

Conclusion

Our present study demonstrates that AMPK exerts dual effects on the PI3K pathway, stimulating PI3K/Akt and inhibiting mTOR/S6K.     

Inhibition of mTORC1 induces loss of E-cadherin through AKT/GSK-3β signaling-mediated upregulation of E-cadherin repressor complexes in non-small cell lung cancer cells

Background

mTOR, which can form mTOR Complex 1 (mTORC1) or mTOR Complex 2 (mTORC2) depending on its binding partners, is frequently deregulated in the pulmonary neoplastic conditions and interstitial lung diseases of the patients treated with rapalogs. In this study, we investigated the relationship between mTOR signaling and epithelial mesenchymal transition (EMT) by dissecting mTOR pathways.

Methods

Components of mTOR signaling pathway were silenced by shRNA in a panel of non-small cell lung cancer cell lines and protein expression of epithelial and mesenchymal markers were evaluated by immunoblotting and immunocytochemistry. mRNA level of the E-cadherin repressor complexes were evaluated by qRT-PCR.

Results

IGF-1 treatment decreased expression of the E-cadherin and rapamycin increased its expression, suggesting hyperactivation of mTOR signaling relates to the loss of E-cadherin. Genetic ablation of rapamycin-insensitive companion of mTOR (Rictor), a component of mTORC2, did not influence E-cadherin expression, whereas genetic ablation of regulatory-associated protein of mTOR (Raptor), a component of mTORC1, led to a decrease in E-cadherin expression at the mRNA level. Increased phosphorylation of AKT at Ser473 and GSK-3β at Ser9 were observed in the Raptor-silenced NSCLC cells. Of the E-cadherin repressor complexes tested, Snail, Zeb2, and Twist1 mRNAs were elevated in raptor-silenced A549 cells, and Zeb2 and Twist1 mRNAs were elevated in Raptor-silenced H2009 cells. These findings were recapitulated by treatment with the GSK-3β inhibitor, LiCl. Raptor knockdown A549 cells showed increased expression of N-cadherin and vimentin with mesenchymal phenotypic changes.

Conclusions

In conclusion, selective inhibition of mTORC1 leads to hyperactivation of the AKT/GSK-3β pathway, inducing E-cadherin repressor complexes and EMT. These findings imply the existence of a feedback inhibition loop of mTORC1 onto mTORC2 that plays a role in the homeostasis of E-cadherin expression and EMT, requiring caution in the clinical use of rapalog and selective mTORC1 inhibitors.     

mTORC2 is a tyrosine kinase

The mammalian target of rapamycin (mTOR), a protein kinase, is the centre of huge attention due to its importance in intracellular signaling and in health and disease. In their recent study, Yin et al. show that mTOR can regulate signaling through the insulin-like growth factor 1 receptor and that it possesses a new enzymatic activity — the ability to phosphorylate proteins on tyrosine residues.mTOR is a large, multi-domain protein; its catalytic domain resembles that of lipid kinases such as phosphoinositide 3-kinase (PI 3-kinase), but mTOR actually has protein kinase activity, adding phosphate groups to serine or threonine residues in a growing catalog of substrates, many of which are involved in anabolic pathways.mTOR binds to several protein partners in the cell to form two distinct types of complexes, termed mTOR complexes 1 and 2 (mTORC1/2¹). These differ in their protein components, substrate specificity and regulation. For example, mTORC1 is activated by amino acids, and by hormones and growth factors. mTORC1 contains a protein termed Raptor which allows it to phosphorylate substrates such as the ribosomal protein S6 kinases (S6Ks), and this effect is blocked by rapamycin.mTORC2 contains Rictor in place of Raptor and therefore phosphorylates a distinct set of substrates. These include regulatory (so-called ''hydrophobic'') sites in a family of protein kinases which include Akt, also called protein kinase B (PKB). Rapamycin does not directly inhibit mTORC2 function, but can impair it after longer-term treatment². The regulation of mTORC2 activity remains poorly understood.mTOR complexes play multifaceted roles in insulin signaling. For example, Akt plays key roles in insulin signaling, mediating the regulation of various proteins involved in the effects of this hormone on metabolism, e.g., glucose transport. Akt signaling indirectly activates mTORC1. In turn, mTORC1 regulates key anabolic processes including protein, lipid and ribosome synthesis. However, mTORC1 can, via the S6Ks, inhibit insulin signaling. This involves the phosphorylation of insulin receptor substrates 1 or 2 (IRS1/2), a crucial link between insulin (and related) receptors and downstream signalling protein, e.g., Akt.The receptors for insulin (InsR) and insulin-like growth factor I (IGF-IR) are ligand-activated tyrosine kinases, which undergo autophosphorylation allowing them to phosphorylate additional proteins such as IRS1. In turn, phosphorylated IRS1 binds PI 3-kinase; this leads to enhanced production of phosphatidylinositol 3,4,5-trisphosphate, PIP₃, and to activation of Akt.Yin et al.³ found that rapamycin led to increased phosphorylation of InsR and IGF-IR at key autophosphorylation sites, reflecting increased kinase activity of these receptors.Knockdown of mTOR or Rictor, or treatment of cells with an inhibitor of mTOR kinase activity, Torin 2, decreased the rapamycin-induced phosphorylation of InsR or IGF-IR, while Raptor knockdown had the converse effect. This indicates the effect requires mTORC2; indeed, the authors show that mTORC2 binds to these receptors, apparently via IRS1/2. However, mTORC2 does not appear to directly phosphorylate IRS1/2. One possible way in which mTORC2 increases tyrosine phosphorylation of InsR or IGF-IR is by stimulating the kinase activity of the receptors which then catalyse the phosphorylation of the receptors on tyrosine. The authors ruled this out, by using kinase-dead versions of the receptors or mTOR. Therefore, mTORC2 promotes the tyrosine phosphorylation of InsR/IGF-1R, which is required for downstream signaling from these receptors. While these authors clearly show that rapamycin causes increased phosphorylation of the mTORC2 substrate AKT, earlier studies showed that, at similar time points of treatment in the same cell-type, rapamycin inhibited AKT phosphorylation indicating interference with mTORC2 function². It is not clear how rapamycin promotes mTORC2 function under the conditions used in this study. Another study⁴ found that mTORC2 promotes degradation of IRS1, suggesting, in contrast to the conclusions of Yin et al., that mTORC2 can promote insulin resistance. These and other data suggest that the web of interactions between these signaling components is indeed very complex (Figure 1).Open in a separate windowFigure 1Summary of the signalling connections discussed here, including the new link described by Yin et al.³ between mTORC2 and the insulin/IGF-1 receptors. Phosphorylation sites are shown schematically (not all are indicated) as ''P'' in a yellow background; Y, S and T indicate tyrosine, serine and threonine respectively. Green and red arrows show activating and inhibitory phosphorylation events respectively. The gray arrow and ''?'' indicate potential further tyrosine phosphorylation events catalysed by mTORC2. Solid arrows show direct phosphorylation events; dashed lines are indirect signalling links.mTOR has previously only been reported to act on serine or threonine residues; the present report shows that mTOR can efficiently phosphorylate tyrosines in vitro using either recombinant InsR or peptides as substrate. These data reveal that mTORC2 function is a ''dual-specificity'' protein kinase phosphorylating tyrosine as well as serine/threonine sites. Interestingly, mTORC1 was unable to phosphorylate tyrosines.Does the mTORC2-stimulated phosphorylation of the InsR/IGF-1R play a role in the actions of the ligands for these receptors? To test this, the authors examined the Rictor knockdown on HepG2 cell proliferation. While this had no effect in the absence of insulin or IGF-1, depletion of Rictor did inhibit proliferation in IGF-1- or insulin-stimulated conditions. Rictor overexpression increased proliferation, an effect that requires the activity of the InsR/IGF-1R.What are the main implications of these data? First, rapamycin may actually promote signaling from the InsR/IGF-1R through mTORC2 (as well as via Grb10, a target for mTORC1 itself^5,6) both by the mechanism delineated here and by abrogating the feedback loop from mTORC1 via the S6Ks to IRS1. Second, combining Ins/IGF-1R receptor inhibitors with mTOR inhibitors may be a more effective anti-cancer treatment than inhibiting the individual pathways. Third, mTORC2 may phosphorylate additional, so far unidentified proteins on tyrosine, adding to the growing repertoire of mTOR substrates.     

Sphingosine kinase 1 expression is regulated by signaling through PI3K, AKT2, and mTOR in human coronary artery smooth muscle cells

Sphingosine kinase 1 (SphK1) is a lipid kinase implicated in mitogenic signaling pathways in vascular smooth muscle cells. We demonstrate that human coronary artery smooth muscle (HCASM) cells require SphK1 for growth and that SphK1 mRNA and protein levels are elevated in PDGF stimulated HCASM cells. To determine the mechanism of PDGF-induced SphK1 expression, we used pharmacological inhibitors of the PI3K/AKT/mTOR signaling pathway. Wortmannin, SH-5, and rapamycin significantly blocked PDGF-stimulated induction of SphK1 mRNA and protein expression, indicating a regulatory role of the PI3K/AKT/mTOR pathway in SphK1 expression. To determine which isoform of AKT regulates SphK1 mRNA and protein levels, siRNAs specific for AKT1, AKT2, and AKT3 were used. We show that AKT2 siRNA significantly blocked PDGF-stimulated increases in SphK1 mRNA and protein expression levels as well as SphK1 enzymatic activity levels. In contrast, AKT1 or AKT3 siRNA did not have an effect. Together, these results demonstrate that the PI3K/AKT/mTOR signaling pathway is involved in regulation of SphK1, with AKT2 playing a key role in PDGF-induced SphK1 expression in HCASM cells.     

Sphingosine kinase 1 expression is regulated by signaling through PI3K, AKT2, and mTOR in human coronary artery smooth muscle cells

Sphingosine kinase 1 (SphK1) is a lipid kinase implicated in mitogenic signaling pathways in vascular smooth muscle cells. We demonstrate that human coronary artery smooth muscle (HCASM) cells require SphK1 for growth and that SphK1 mRNA and protein levels are elevated in PDGF stimulated HCASM cells. To determine the mechanism of PDGF-induced SphK1 expression, we used pharmacological inhibitors of the PI3K/AKT/mTOR signaling pathway. Wortmannin, SH-5, and rapamycin significantly blocked PDGF-stimulated induction of SphK1 mRNA and protein expression, indicating a regulatory role of the PI3K/AKT/mTOR pathway in SphK1 expression. To determine which isoform of AKT regulates SphK1 mRNA and protein levels, siRNAs specific for AKT1, AKT2, and AKT3 were used. We show that AKT2 siRNA significantly blocked PDGF-stimulated increases in SphK1 mRNA and protein expression levels as well as SphK1 enzymatic activity levels. In contrast, AKT1 or AKT3 siRNA did not have an effect. Together, these results demonstrate that the PI3K/AKT/mTOR signaling pathway is involved in regulation of SphK1, with AKT2 playing a key role in PDGF-induced SphK1 expression in HCASM cells.     

8-Chloroadenosine Sensitivity in Renal Cell Carcinoma Is Associated with AMPK Activation and mTOR Pathway Inhibition

The adenosine analog 8-chloroadenosine has been shown to deplete ATP and inhibit tumor growth in hematological malignancies as well as in lung and breast cancer cell lines. We investigated effects of 8-chloroadenosine on clear cell (cc) renal cell carcinoma (RCC) cell lines. 8-chloroadenosine was effective against ccRCC cell viability in vitro, with IC₅₀ ranging from 2 μM in the most sensitive CAKI-1 to 36 μM in the most resistant RXF-393. Proteomic analysis by reverse-phase protein array revealed that 8-chloroadenosine treatment leads to inhibition of the mTOR pathway. In time-course experiments, 8-chloroadenosine treatment rapidly activated AMPK, measured by AMPK and ACC phosphorylation, and subsequently caused dephosphorylation of p70S6K and ribosomal protein RPS6 in the sensitive cell lines. However, in the resistant cell lines, AMPK activity and the mTOR pathway were unaffected by the treatment. We also noted that the resistant cell lines had elevated basal levels of phospho RPS6 and AKT. Inhibition of PI3K pathway enhanced the efficacy of 8-chloroadenosine across all cell lines. Our observations indicate that 8-chloroadenosine activity is associated with inhibition of the mTOR pathway, and that phospho RPS6 and PI3K pathway activation status may determine resistance. Among solid tumors, RCC is one of the few susceptible to mTOR inhibition. We thus infer that 8-chloroadenosine may be effective in RCC by activating AMPK and inhibiting the mTOR pathway.     

Insulin Receptor Substrate 1, the Hub Linking Follicle-stimulating Hormone to Phosphatidylinositol 3-Kinase Activation

The ubiquitous phosphatidylinositol 3-kinase (PI3K) signaling pathway regulates many cellular functions. However, the mechanism by which G protein-coupled receptors (GPCRs) signal to activate PI3K is poorly understood. We have used ovarian granulosa cells as a model to investigate this pathway, based on evidence that the GPCR agonist follicle-stimulating hormone (FSH) promotes the protein kinase A (PKA)-dependent phosphorylation of insulin receptor substrate 1 (IRS1) on tyrosine residues that activate PI3K. We report that in the absence of FSH, granulosa cells secrete a subthreshold concentration of insulin-like growth factor-1 (IGF-1) that primes the IGF-1 receptor (IGF-1R) but fails to promote tyrosine phosphorylation of IRS1. FSH via PKA acts to sensitize IRS1 to the tyrosine kinase activity of the IGF-1R by activating protein phosphatase 1 (PP1) to promote dephosphorylation of inhibitory Ser/Thr residues on IRS1, including Ser⁷⁸⁹. Knockdown of PP1β blocks the ability of FSH to activate PI3K in the presence of endogenous IGF-1. Activation of PI3K thus requires both PKA-mediated relief of IRS1 inhibition and IGF-1R-dependent tyrosine phosphorylation of IRS1. Treatment with FSH and increasing concentrations of exogenous IGF-1 triggers synergistic IRS1 tyrosine phosphorylation at PI3K-activating residues that persists downstream through protein kinase B (AKT) and FOXO1 (forkhead box protein O1) to drive synergistic expression of genes that underlies follicle maturation. Based on the ability of GPCR agonists to synergize with IGFs to enhance gene expression in other cell types, PP1 activation to relieve IRS1 inhibition may be a more general mechanism by which GPCRs act with the IGF-1R to activate PI3K/AKT.     

Rapamycin Inhibits IGF-1-Mediated Up-Regulation of MDM2 and Sensitizes Cancer Cells to Chemotherapy

The Murine Double Minute 2 (MDM2) protein is a key regulator of cell proliferation and apoptosis that acts primarily by inhibiting the p53 tumor suppressor. Similarly, the PI3-Kinase (PI3K)/AKT pathway is critical for growth factor-mediated cell survival. Additionally, it has been reported that AKT can directly phosphorylate and activate MDM2. In this study, we show that IGF-1 up-regulates MDM2 protein levels in a PI3K/AKT-dependent manner. Inhibition of mTOR by rapamycin or expression of a dominant negative eukaryotic initiation factor 4E binding protein 1 (4EBP1) mutant protein, as well as ablation of eukaryotic initiation factor 4E (eIF4E), efficiently abolishes IGF-1-mediated up-regulation of MDM2. In addition, we show that rapamycin effectively inhibits MDM2 expression and sensitizes cancer cells to chemotherapy. Taken together, this study reveals a novel mechanism by which IGF-1 activates MDM2 via the mTOR pathway, and that pharmacologic inhibition of mTOR combined with chemotherapy may be more effective in treatment of a subset of cancers harboring increased MDM2 activation.     

四物汤在卵巢衰老大鼠骨骼肌中的雌激素样作用机制研究

摘要 目的：探讨四物汤通过胰岛素样生长因子-1（Insulin-ike growth factors-1，IGF-1）/磷脂酰肌醇3-激酶（PI3K）/蛋白激酶B（AKT）/雷帕霉素靶蛋白（Mammalian target of rapamycin，mTOR）mTOR信号通路发挥对4-乙烯基环己烯二环氧化合物（4-Vinylcyclohexene Diepoxide，VCD）诱导的卵巢衰老大鼠骨骼肌的保护作用及其分子机制。方法：选用28日龄雌性F-344大鼠，随机选取6只作为空白对照组，剩余大鼠连续腹腔注射VCD溶液（160 mg/kg/d）20天，每天阴道涂片检测动情周期，连续观察12 d无角化细胞或仅有少量角化细胞即符合"卵巢衰老"表现，经动情周期筛选出24只造模成功的大鼠，分为模型组（Model）、阳性（戊酸雌二醇，E₂）对照组、四物汤高剂量（SWT-H）组和四物汤低剂量（SWT-L）组，每组6只，灌胃持续3周后取材。取大鼠骨骼肌组织进行HE染色观察病理组织结构；MASSON染色骨骼肌纤维形态变化；RT-PCR检测骨骼肌组织中各基因的mRNA水平。结果：与空白组比较，模型组骨骼肌肌纤维排列疏松，分布不均且有断点，胞浆不均，细胞核增多，出现增生的结缔组织，胶原纤维逐渐增多且肌纤维间距变宽；与模型组相比，E₂组、SWT-H组与SWT-L组肌纤维排列相对整齐，胞浆较为均匀。肌肉形态较规则，纤维间距缩短，胶原纤维减少。RT-PCR结果显示，与空白组相比，模型组IGF-1、PI3K、AKT、mTOR基因的mRNA表达量明显下调，E₂组、SWT-H组与SWT-L组的IGF-1、PI3K、mTOR的表达明显上升，SWT-H组AKT表达无明显变化，无统计学意义，SWT-L组AKT表达相对降低。与模型组相比，E₂组、SWT-H组与SWT-L组的IGF-1、AKT、mTOR的表达明显增加，且具有剂量依赖性，PI3K表达增加，但无明显剂量依赖性。结论：四物汤可以明显改善VCD诱导的卵巢衰老大鼠的肌肉减少情况，其机制可能是通过IGF-1-PI3K-AKT-mTOR信号通路来发挥其抑制肌肉流失的作用。     

Propofol alleviates hypoxia-induced nerve injury in PC-12 cells by up-regulation of microRNA-153

Background

Although the neuroprotective role of propofol has been identified recently, the regulatory mechanism associated with microRNAs (miRNAs/miRs) in neuronal cells remains to be poorly understood. We aimed to explore the regulatory mechanism of propofol in hypoxia-injured rat pheochromocytoma (PC-12) cells.

Methods

PC-12 cells were exposed to hypoxia, and cell viability and apoptosis were assessed by CCK-8 assay and flow cytometry assay/Western blot analysis, respectively. Effects of propofol on hypoxia-injured cells were measured, and the expression of miR-153 was determined by stem-loop RT-PCR. After that, whether propofol affected PC-12 cells under hypoxia via miR-153 was verified, and the downstream protein of miR-153 as well as the involved signaling cascade was finally explored.

Results

Hypoxia-induced decrease of cell viability and increase of apoptosis were attenuated by propofol. Then, we found hypoxia exposure up-regulated miR-153 expression, and the level of miR-153 was further elevated by propofol in hypoxia-injured PC-12 cells. Following experiments showed miR-153 inhibition reversed the effects of propofol on hypoxia-treated PC-12 cells. Afterwards, we found BTG3 expression was negatively regulated by miR-153 expression, and BTG3 overexpression inhibited the mTOR pathway and AMPK activation. Besides, hypoxia inhibited the mTOR pathway and AMPK, and these inhibitory effects could be attenuated by propofol.

Conclusion

Propofol protected hypoxia-injured PC-12 cells through miR-153-mediataed down-regulation of BTG3. BTG3 could inhibit the mTOR pathway and AMPK activation.

     

High nutrient intake during the early postnatal period accelerates skeletal muscle fiber growth and maturity in intrauterine growth-restricted pigs

Background

Intrauterine growth-restricted (IUGR) neonates impair postnatal skeletal muscle growth. The aim of this study was to investigate whether high nutrient intake (HNI) during the suckling period could improve muscle growth and metabolic status of IUGR pigs.

Methods

Twelve pairs of IUGR and normal birth weight (NBW) pigs (7 days old) were randomly assigned to adequate nutrient intake and HNI formula milk groups. Psoas major (PM) muscle sample was obtained after 21 days of rearing.

Results

IUGR decreased cross-sectional areas (CSA) and myofiber numbers, activity of lactate dehydrogenase (LDH), and mRNA expression of insulin-like growth factor 1 (IGF-1), IGF-1 receptor (IGF-1R), mammalian target of rapamycin (mTOR), ribosomal protein s6 (RPS6), eukaryotic translation initiation factor 4E (eIF4E), protein expression of phosphorylated mTOR (P-mTOR), and phosphorylated protein kinase B (P-Akt) in the PM muscle of pigs. Irrespective of birth weight, HNI increased muscle weight and CSA, the concentration of RNA, and ratio of RNA to DNA, as well as ratio of LDH to β-hydroxy-acyl-CoA-dehydrogenase in the PM muscle of pigs. Furthermore, HNI increased percentages of MyHC IIb, mRNA expression of IGF-1, IGF-1R, Akt, mTOR, RPS6, and eIF4E, as well as protein expression of P-mTOR, P-Akt, P-RPS6, and P-eIF4E in the PM muscle of pigs.

Conclusion

The present findings suggest that high nutrient intake during the suckling period could improve skeletal muscle growth and maturity, which is associated with increasing the expression of protein deposition-related genes and accelerating the development of glycolytic-type myofiber in pigs.

     

FoxP3-miR-150-5p/3p suppresses ovarian tumorigenesis via an IGF1R/IRS1 pathway feedback loop

Ovarian cancer (OC) causes more deaths than any other gynecological cancer. Many cellular pathways have been elucidated to be associated with OC development and progression. Specifically, the insulin-like growth factor 1 receptor/insulin receptor substrate 1 (IGF1R/IRS1) pathway participates in OC development. Moreover, accumulating evidence has shown that microRNA deregulation contributes to tumor initiation and progression. Here, our study aimed to investigate the molecular functions and regulatory mechanisms of miR-150, specifically, in OC. We found that the expression of miR-150-5p/3p and their precursor, mir-150, was downregulated in OC tissues; lower mir-150 levels were associated with poor OC patient outcomes. Ectopic mir-150 expression inhibited OC cell growth and metastasis in vitro and in vivo. Furthermore, both IRS1 and IGF1R were confirmed as direct targets of miR-150-5p/3p, and the miR-150-IGF1R/IRS1 axis exerted antitumor effects via the PI3K/AKT/mTOR pathway. Forkhead box protein 3 (FoxP3) positively regulated the expression of miR-150-5p/3p by binding to the mir-150 promoter. In turn, the PI3K/AKT/mTOR pathway downregulated FoxP3 and miR-150-5p/3p. Taken together, these findings indicate that a complex FoxP3-miR-150-IGF1R/IRS1-PI3K/AKT/mTOR feedback loop regulates OC pathogenesis, providing a novel mechanism for miR-150 as a tumor suppressor miRNA in OC.Subject terms: Cancer, Translational research     

Effect of combined aerobic and strength exercises on the regulation of mitochondrial biogenesis and protein synthesis and degradation in human skeletal muscle

We tested the hypothesis that strength exercise after intermittent aerobic exercise might activate signaling pathways that regulate mitochondrial biogenesis (activation of the AMPK and p38 pathways; the expression of PGC-1α, NT-PGC-1α, TFAM, and VEGFA mRNA), protein synthesis (phosphorylation level of p70S6K1^Thr389 and eEF2^Thr56; the expression IGF-1Ea, IGF-1Ec (MGF), and REDD1 mRNA) and proteolysis (phosphorylation level of FOXO1^Ser256; the expression of MURF1, MAFbx, and Myostatin mRNA) in trained skeletal muscles. Nine amateur endurance-trained athletes performed an intermittent aerobic cycling (70 min), followed by one-leg strength exercise (ES: four sets of knee extensions till exhaustion), while the other leg was resting (E). Gene expression and protein level were evaluated in samples from m. vastus lateralis taken before the exercise, 40 min, 5 and 22 h after the aerobic exercise. The phosphorylation level of the АСС^Ser79/222 (an endogenous marker of AMPK activity) and the expression of PGC-1α-related gene TFAM (a marker of mitochondrial biogenesis) were increased after E exercise and did not changed after ES exercise. The expression of PGC-1α and truncated isoform NT-PGC-1α was increased in both legs as well. Insulin concentration in blood was decreased significantly (7.5-fold) after aerobic exercise; the phosphorylation level of FOXO^Ser256 (a regulator of ubiquitin-related proteolysis) was decreased in both legs, which means that it was activated in both types of exercises; at the same time, the expression of the E3-ubiquitin ligase gene MURF1, its target, was only increased after E exercise. Neither aerobic or combined exercise had a significant effect on the regulation of protein synthesis: there were no changes in either expression of IGF-1Ea and IGF-1Ec(MGF) mRNA isoforms or the phosphorylation levels of markers of protein synthesis p70S6K1^Thr389 and eEF2^Thr56. Thus, the performance of strength exercise immediately after aerobic one prevented the activation of mitochondrial biogenesis in endurance-trained muscles: activation of AMPK pathway and the expression of TFAM are decreased, while protein synthesis regulation is not affected. At the same time, the strength exercise inhibited the expression of MURF1 gene (a marker of ubiquitin proteasome system), which was induced by aerobic exercise. We suggest that strength exercise performed immediately after intense intermittent aerobic exercise may have a negative effect on aerobic performance if used chronically.     

Differential regulation of AMP-activated kinase and AKT kinase in response to oxygen availability in crucian carp (Carassius carassius)

We investigated whether two kinases critical for survival during periods of energy deficiency in anoxia-intolerant mammalian species, AMP-activated kinase (AMPK), and protein kinase B (AKT), are equally important for hypoxic/anoxic survival in the extremely anoxia-tolerant crucian carp (Carassius carassius). We report that phosphorylation of AMPK and AKT in heart and brain showed small changes after 10 days of severe hypoxia (0.3 mg O2/l at 9 degrees C). In contrast, anoxia exposure (0.01 mg O2/l at 8 degrees C) substantially increased AMPK phosphorylation but decreased AKT phosphorylation in carp heart and brain, indicating activation of AMPK and deactivation of AKT. In agreement, blocking the activity of AMPK in anoxic fish in vivo with 20 mg/kg Compound C resulted in an elevated metabolic rate (as indicated by increased ethanol production) and tended to reduce energy charge. This is the first in vivo experiment with Compound C in a nonmammalian vertebrate, and it appears that AMPK plays a role in mediating anoxic metabolic depression in crucian carp. Real-time RT-PCR analysis of the investigated AMPK subunit revealed that the most likely composition of subunits in the carp heart is alpha2, beta1B, gamma2a, whereas a more even expression of subunits was found in the brain. In the heart, expression of the regulatory gamma2-subunit increased in the heart during anoxia. In the brain, expression of the alpha1-, alpha2-, and gamma1-subunits decreased with anoxia exposure, but expression of the gamma2-subunit remained constant. Combined, our findings suggest that AMPK and AKT may play important, but opposing roles for hypoxic/anoxic survival in the anoxia-tolerant crucian carp.     

Sestrin2 modulates AMPK subunit expression and its response to ionizing radiation in breast cancer cells

Background

The sestrin family of stress-responsive genes (SESN1-3) are suggested to be involved in regulation of metabolism and aging through modulation of the AMPK-mTOR pathway. AMP-activated protein kinase (AMPK) is an effector of the tumour suppressor LKB1, which regulates energy homeostasis, cell polarity, and the cell cycle. SESN1/2 can interact directly with AMPK in response to stress to maintain genomic integrity and suppress tumorigenesis. Ionizing radiation (IR), a widely used cancer therapy, is known to increase sestrin expression, and acutely activate AMPK. However, the regulation of AMPK expression by sestrins in response to IR has not been studied in depth.

Methods and Findings

Through immunoprecipitation we observed that SESN2 directly interacted with the AMPKα1β1γ1 trimer and its upstream regulator LKB1 in MCF7 breast cancer cells. SESN2 overexpression was achieved using a Flag-tagged SESN2 expression vector or a stably-integrated tetracycline-inducible system, which also increased AMPKα1 and AMPKβ1 subunit phosphorylation, and co-localized with phosphorylated AMPKα-Thr127 in the cytoplasm. Furthermore, enhanced SESN2 expression increased protein levels of LKB1 and AMPKα1β1γ1, as well as mRNA levels of LKB1, AMPKα1, and AMPKβ1. Treatment of MCF7 cells with IR elevated AMPK expression and activity, but this effect was attenuated in the presence of SESN2 siRNA. In addition, elevated SESN2 inhibited IR-induced mTOR signalling and sensitized MCF7 cells to IR through an AMPK-dependent mechanism.

Conclusions

Our results suggest that in breast cancer cells SESN2 is associated with AMPK, it is involved in regulation of basal and IR-induced expression and activation of this enzyme, and it mediates sensitization of cancer cells to IR.     

Activation of adenosine monophosphate activated protein kinase inhibits growth of multiple myeloma cells

The role of adenosine monophosphate activated protein kinase (AMPK) in regulating multiple myeloma (MM) cell growth is not yet clear. In this study, we show that the AMPK activators 5-aminoimidazole-4-carboxamide riboside (AICAr) and D942 inhibit cell growth in MM cell lines. AICAr also induced an S-phase cell cycle arrest in all four tested cell lines and led to phosphorylation and thus activation of AMPK. Furthermore, the inhibition of a nucleoside transporter by nitrobenzyl-thio-9-beta-d-ribofuranosylpurine (NBTI), inhibition of the adenosine kinase by iodotubericidine and inhibition of AMPK by AMPKI Compound C reversed AICAr effects, indicating that the cellular effects of AICAr were mediated by AMPK. Activation of AMPK inhibited basal extracellular signal-regulated kinase (ERK), mammalian target of rapamycin (mTOR) and P70S6 kinase (P70S6K) as well as AKT phosphorylation, and blocked IL-6, IGF-1, and HS-5 stromal cell conditioned medium-induced increase of cell growth. Troglitazone, which has previously been shown to activate AMPK, similarly inhibited MM cell growth, activated AMPK, and decreased ERK and P70S6K phosphorylation. Our results suggest that activation of AMPK inhibits MM cell growth despite stimulation with IL-6, IGF-1, or HS-5 stromal cell conditioned medium and represents a potential new target in the therapy of MM.     

Resistin promotes cardiac hypertrophy via the AMP-activated protein kinase/mammalian target of rapamycin (AMPK/mTOR) and c-Jun N-terminal kinase/insulin receptor substrate 1 (JNK/IRS1) pathways

Resistin has been suggested to be involved in the development of diabetes and insulin resistance. We recently reported that resistin is expressed in diabetic hearts and promotes cardiac hypertrophy; however, the mechanisms underlying this process are currently unknown. Therefore, we wanted to elucidate the mechanisms associated with resistin-induced cardiac hypertrophy and myocardial insulin resistance. Overexpression of resistin using adenoviral vector in neonatal rat ventricular myocytes was associated with inhibition of AMP-activated protein kinase (AMPK) activity, activation of tuberous sclerosis complex 2/mammalian target of rapamycin (mTOR) pathway, and increased cell size, [(3)H]leucine incorporation (i.e. protein synthesis) and mRNA expression of the hypertrophic marker genes, atrial natriuretic factor, brain natriuretic peptide, and β-myosin heavy chain. Activation of AMPK with 5-aminoimidazole-4-carbozamide-1-β-D-ribifuranoside or inhibition of mTOR with rapamycin or mTOR siRNA attenuated these resistin-induced changes. Furthermore, resistin increased serine phosphorylation of insulin receptor substrate (IRS1) through the activation of the apoptosis signal-regulating kinase 1/c-Jun N-terminal Kinase (JNK) pathway, a module known to stimulate insulin resistance. Inhibition of JNK (with JNK inhibitor SP600125 or using dominant-negative JNK) reduced serine 307 phosphorylation of IRS1. Resistin also stimulated the activation of p70(S6K), a downstream kinase target of mTOR, and increased phosphorylation of the IRS1 serine 636/639 residues, whereas treatment with rapamycin reduced the phosphorylation of these residues. Interestingly, these in vitro signaling pathways were also operative in vivo in ventricular tissues from adult rat hearts overexpressing resistin. These data demonstrate that resistin induces cardiac hypertrophy and myocardial insulin resistance, possibly via the AMPK/mTOR/p70(S6K) and apoptosis signal-regulating kinase 1/JNK/IRS1 pathways.