The Autophagy-Related Protein GABARAP Is Induced during Overwintering in the Bean Bug (Hemiptera: Alydidae).

Anautogeny is a reproductive strategy by which females do not reproduce until they feed. Therefore, nutritional signals must inform the reproductive tissues, and cells that the organism has reached a nutritional status suitable for triggering reproductive processes. One of the possible pathways involved in anautogeny is the "target of rapamycin" (TOR) pathway, which has been described as connecting the nutritional status with growth, proliferation, and cancer. The German cockroach, Blattella germanica, is an anautogenous species whose vitellogenesis is governed by juvenile hormone. In the present report, we describe the cloning of TOR cDNA from B. germanica (BgTOR). Expression studies showed that BgTOR is expressed in adult female corpora allata and fat body. BgTOR knockdown using systemic RNAi in vivo produced a severe inhibition of juvenile hormone synthesis in adult female corpora allata, together with a reduction of mRNA levels corresponding to 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase-1, HMG-CoA synthase-2, and HMG-CoA reductase. In addition, there was a reduction of vitellogenin mRNA in the fat body, and ovaries did not grow. Analysis of TOR expression in corpora allata of fed and starved females suggested that TOR is not regulated at the transcriptional level. Nevertheless, there was a reduction in HMG-CoA synthases and reductase mRNA in corpora allata (but not in the fat body) of starved females, together with a dramatic reduction of juvenile hormone production and ovary development. Taken together, our results indicate that TOR knockdown mimics starvation in terms of corpora allata activity, and suggest that nutritional signals that activate juvenile hormone biosynthesis and vitellogenin production are mediated by the TOR pathway.

Sestrins (Sesns) are a family of highly conserved stress-responsive proteins, transcriptionally regulated by p53 and forkhead transcription factor that exhibit oxidoreductase activity in vitro and can protect cells from oxidative stress. However, their major biochemical and physiological function does not appear to depend on their redox (reduction and oxidation) activity. Sesns promote activation of adenosine-5′-monophosphate (AMP)-dependent protein kinase in both mammals and flies. Stress-induced Sesn expression results in inhibition of the target of rapamycin complex 1 (TORC1) and the physiological and pathological implications of disrupting the Sesns-TORC1 crosstalk are now being unravelled. Detailing their mechanism of action and exploring their roles in human physiology point to exciting new insights to topics as diverse as stress, cancer, metabolism and aging.

Objective To study the role of mTOR - autophagy signaling pathway in diabetic nephropathy of rats. Methods The SD rats were divided into the normal group and the control group, and the control group was constructed type 2 diabetic nephropathy model with streptozocin. 8 weeks later, we tested body weight, kidney weight, urine trace albumin, blood biochemical item of the rats. Glomerulus and renal tubule were observed by light microscopy. The renal LC3A/B and p-S6 expression were tested by Western blot. Results Diabetic rats model was established successfully. We found weight loss, kidney weight increase, blood sugar, triglyceride, and urea nitrogen increased, but the cholesterol and albumin decreased, urine trace albumin increased in the control group; the glomerulus hypertrophied, mesangial membrane cell proliferation and mesangial matrix increased; Western blot found that kidney tissues autophagy activity of diabetic rats significantly decreased as the LC3A/B decreased; mTOR was activated, because the p-S6 level increased. Conclusions mTOR activation and autophagy ability decline are found in the diabetic nephropathy, mTOR - autophagy signaling pathway may be one of the pathogenesis of diabetic nephropathy. Key words: Diabetic nephropathy; mTOR; Autophagy

metabolic syndrome is a cluster of conditions including insulin resistance, dyslipidemia, hypertension, and central obesity, and it results in an increased risk of type 2 diabetes mellitus and cardiovascular diseases such as atherosclerosis. These conditions are rising year to year and are a leading

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that plays an essential role in cell growth control. mTOR stimulates cell growth by phosphorylating p70 ribosomal S6 kinase (S6K) and eukaryote initiation factor 4E-binding protein 1 (4EBP1). The mTOR pathway is regulated by a wide variety of cellular signals, including mitogenic growth factors, nutrients, cellular energy levels, and stress conditions. Recent studies have proposed several mechanisms to explain how mTOR is regulated by growth factors and cellular energy levels. However, little is known as to how mTOR is regulated by stress conditions. We observed that two stress-induced proteins, RTP801/Redd1 and RTP801L/Redd2, potently inhibit signaling through mTOR. Our data support that RTP801 and RTP801L work downstream of AKT and upstream of TSC2 to inhibit mTOR functions. These results add a new dimension to mTOR pathway regulation and provide a possible molecular mechanism of how cellular stress conditions may regulate mTOR function.

None for Perspective.

THERAPEUTIC EFFICACY OF COMBINATION OF MTOR INHIBITORS AND AMPK ACTIVATORS IN NON-SMALL CELL LUNG CANCER. By Grinal Michael Corriea, Master of Science. A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science at Virginia Commonwealth University. Virginia Commonwealth University, 2014 Major Director: Richard G. Moran, PhD, Professor, Department of Pharmacology & Toxicology Pemetrexed (PTX), an antifolate drug, has been approved by the US FDA for first line therapy of mesothelioma and non-small cell lung cancer. In addition to its primary site of action on thymidylate synthase (TS), PTX also inhibits the second folate-dependent enzyme of purine biosynthesis aminoimidazolecarboxamide ribonucleotide formyltransferase (AICART). The accumulation of the substrate for AICART, ZMP, in PTX-inhibited cancer cells leads to activation of AMP-activated protein kinase (AMPK) with subsequent inhibition of mammalian target of rapamycin (mTOR) and hypophosphorylation of its downstream targets responsible for protein synthesis and cell proliferation. Inhibitors of mTORC1 like Rapamycin and its analogs (rapalogs) have only partial effects on tumor cells as they do not inhibit mTORC2, which phosphorylates Akt subsequently relieving the inhibition of mTORC1, thus leading to poor cytotoxicity by rapalogs. AMPK exerts control on mTORC1 kinase activity and PTX mediated activation of AMPK leads to its subsequent downregulation and hence, would be expected to have a therapeutic interaction with direct mTOR inhibitors. AZD8055, an ATPcompetitive inhibitor of mTOR kinase, potently inhibits both mTORC1 and mTORC2 and therefore, can overcome the feedback mechanism(s) limiting the action of rapalogs to cytostatic effects. To study the effects of AMPK activation and mTOR inhibition pharmacologically, we performed growth suppression assays using pemetrexed, AICAR, RAD001, and AZD8055. The effect of inhibition of mTOR with these drugs was assessed by examining the dephosphorylation of mTORC1 substrates S6K1 and 4E-BP1, as single agents and in combination, at their 50% inhibitory concentrations (IC50) by western blotting. Our data suggested that AMPK activation via PTX mediated AICART inhibition in combination with direct mTOR inhibition by AZD8055 has a synergistic interaction on the proliferation of NSCLC cells in culture. Inhibition of mTOR endogenously by pemetrexed, along with direct pharmacological inhibition of mTOR prevents the feedback circuit which may compromise the therapeutic efficacy of rapamycin analogs. Pemetrexed and AZD8055, as single agents, demonstrated inhibitory activity on phosphorylation events of mTORC1 substrates. This activity was markedly increased by combining both the drugs. Our findings suggest that direct inhibitors of mTOR enhance the effects of activators of AMPK. These effects appear to be mediated via combined effects on mTORC1. Taken together, the combination of catalytic site mTOR inhibitors and pemetrexed is a promising therapeutic strategy and calls for further preclinical and clinical investigations.

O-linked N-acetylglucosamine, better known as O-GlcNAc, is a sugar post-translational modification participating in a diverse range of cell functions. Disruptions in the cycling of O-GlcNAc mediated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively, is a driving force for aberrant cell signaling in disease pathologies, such as diabetes, obesity, Alzheimer's disease, and cancer. Production of UDP-GlcNAc, the metabolic substrate for OGT, by the Hexosamine Biosynthetic Pathway (HBP) is controlled by the input of amino acids, fats, and nucleic acids, making O-GlcNAc a key nutrient-sensor for fluctuations in these macromolecules. The mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) pathways also participate in nutrient-sensing as a means of controlling cell activity and are significant factors in a variety of pathologies. Research into the individual nutrient-sensitivities of the HBP, AMPK, and mTOR pathways has revealed a complex regulatory dynamic, where their unique responses to macromolecule levels coordinate cell behavior. Importantly, cross-talk between these pathways fine-tunes the cellular response to nutrients. Strong evidence demonstrates that AMPK negatively regulates the mTOR pathway, but O-GlcNAcylation of AMPK lowers enzymatic activity and promotes growth. On the other hand, AMPK can phosphorylate OGT leading to changes in OGT function. Complex sets of interactions between the HBP, AMPK, and mTOR pathways integrate nutritional signals to respond to changes in the environment. In particular, examining these relationships using systems biology approaches might prove a useful method of exploring the complex nature of cell signaling. Overall, understanding the complex interactions of these nutrient pathways will provide novel mechanistic information into how nutrients influence health and disease.

iPSCNPC transplantation alleviates brain injury after intracerebral hemorrhage in mice by downregulating autophagy via AMPK/ mTOR signaling pathway.

To investigate the effect of emodin on high glucose (HG)-induced podocyte apoptosis and whether the potential anti-apoptotic mechanism of emodin is related to induction of adenosine-monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR)-mediated autophagy in podocytes (MPC5 cells) in vitro. MPC5 cells were treated with different concentrations of HG (2.5, 5, 10, 20, 40, 80 and 160 mmol/L), emodin (2, 4, 8 µ mol/L), or HG (40 mmol/L) and emodin (4 µ mol/L) with or without rapamycin (Rap, 100 nmol/L) and compound C (10 µ mol/L). The viability and apoptosis of MPC5 cells were detected using cell counting kit-8 (CCK-8) assay and flow cytometry analysis, respectively. The expression levels of cleaved caspase-3, autophagy marker light chain 3 (LC3) I/II, and AMPK/mTOR signaling pathway-related proteins were determined by Western blot. The changes of morphology and RFP-LC3 fluorescence were observed under microscopy. HG at 20, 40, 80 and 160 mmol/L dose-dependently induced cell apoptosis in MPC5 cells, whereas emodin (4 µ mol/L) significantly ameliorated HG-induced cell apoptosis and caspase-3 cleavage (P<0.01). Emodin (4 µ mol/L) significantly increased LC3-II protein expression levels and induced RFP-LC3-containing punctate structures in MPC5 cells (P<0.01). Furthermore, the protective effects of emodin were mimicked by rapamycin (100 nmol/L). Moreover, emodin increased the phosphorylation of AMPK and suppressed the phosphorylation of mTOR. The AMPK inhibitor compound C (10 µ mol/L) reversed emodin-induced autophagy activation. Emodin ameliorated HG-induced apoptosis of MPC5 cells in vitro that involved induction of autophagy through the AMPK/mTOR signaling pathway, which might provide a potential therapeutic option for diabetic nephropathy.

Targeting of AMPK/MTOR signaling in the management of atherosclerosis: Outmost leveraging.

Birt-Hogg-Dubé syndrome, a hamartoma disorder characterized by benign tumors of the hair follicle, lung cysts, and renal neoplasia, is caused by germ-line mutations in the BHD(FLCN) gene, which encodes a tumor-suppressor protein, folliculin (FLCN), with unknown function. The tumor-suppressor proteins encoded by genes responsible for several other hamartoma syndromes, LKB1, TSC1/2, and PTEN, have been shown to be involved in the mammalian target of rapamycin (mTOR) signaling pathway. Here, we report the identification of the FLCN-interacting protein, FNIP1, and demonstrate its interaction with 5' AMP-activated protein kinase (AMPK), a key molecule for energy sensing that negatively regulates mTOR activity. FNIP1 was phosphorylated by AMPK, and its phosphorylation was reduced by AMPK inhibitors, which resulted in reduced FNIP1 expression. AMPK inhibitors also reduced FLCN phosphorylation. Moreover, FLCN phosphorylation was diminished by rapamycin and amino acid starvation and facilitated by FNIP1 overexpression, suggesting that FLCN may be regulated by mTOR and AMPK signaling. Our data suggest that FLCN, mutated in Birt-Hogg-Dubé syndrome, and its interacting partner FNIP1 may be involved in energy and/or nutrient sensing through the AMPK and mTOR signaling pathways.

BackgroundHepatocellular carcinoma (HCC) is one of the most common malignancies around the world. It has been verified that the expression of SOX18 is correlated to poor clinical prognosis in patients with ovarian cancer, non-small cell lung cancer, or breast invasive ductal carcinoma. However, the expression pattern and biological function of SOX18 in HCC tissues remains unclear. In this study, we set out to investigate the associated biological function and potential molecular mechanism of the SOX18 gene in HCC cells.Material/MethodsThe mRNA and protein expression levels of experimental related genes were detected by real-time polymerase chain reaction and western blotting assay, respectively. The MTT method was used to assess cell viability, and cell apoptosis analysis was performed by means of FACScan flow cytometry. Wound-healing assay and Transwell analysis were performed to evaluate the ability of cell migration and invasiveness, respectively.ResultsSOX18 was highly expressed in various HCC cell lines. In addition, SOX18 promoted cell viability, migration, and invasion and simultaneously induce cell apoptosis. SOX18 promoted epithelial-to-mesenchymal transition (EMT) progression, and SOX18 downregulation activated the autophagy signaling pathway AMPK/mTOR in HCC cells.ConclusionsSOX18 downregulation in HCC cells suppressed cell viability and metastasis, induced cell apoptosis and hindered the occurrence and progression of tumor cells by participating in the EMT process and regulating the autophagy signaling pathway AMPK/mTOR.

Amino acids regulate energy utilization through mammalian target of rapamycin complex 1 and adenosine monophosphate activated protein kinase pathway in porcine enterocytes.

Amino acids positively regulate signaling through the mammalian target of rapamycin (mTOR). Recent work demonstrated the importance of the tuberous sclerosis protein TSC2 for regulation of mTOR by insulin. TSC2 contains a GTPase-activator domain that promotes hydrolysis of GTP bound to Rheb, which positively regulates mTOR signaling. Some studies have suggested that TSC2 also mediates the control of mTOR by amino acids. In cells lacking TSC2, amino acid withdrawal still results in dephosphorylation of S6K1, ribosomal protein S6, the eukaryotic initiation factor 4E-binding protein, and elongation factor-2 kinase. The effects of amino acid withdrawal are diminished by inhibiting protein synthesis or adding back amino acids. These studies demonstrate that amino acid signaling to mTOR occurs independently of TSC2 and involves additional unidentified inputs. Although TSC2 is not required for amino acid control of mTOR, amino acid withdrawal does decrease the proportion of Rheb in the active GTP-bound state. Here we also show that Rheb and mTOR form stable complexes, which are not, however, disrupted by amino acid withdrawal. Mutants of Rheb that cannot bind GTP or GDP can interact with mTOR complexes. We also show that the effects of hydrogen peroxide and sorbitol, cell stresses that impair mTOR signaling, are independent of TSC2. Finally, we show that the ability of energy depletion (which impairs mTOR signaling in TSC2+/+ cells) to increase the phosphorylation of eukaryotic elongation factor 2 is also independent of TSC2. This likely involves the phosphorylation of the elongation factor-2 kinase by the AMP-activated protein kinase.