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      ER Stress Induces Anabolic Resistance in Muscle Cells through PKB-Induced Blockade of mTORC1

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          Abstract

          Background

          Anabolic resistance is the inability to increase protein synthesis in response to an increase in amino acids following a meal. One potential mediator of anabolic resistance is endoplasmic reticulum (ER) stress. The purpose of the present study was to test whether ER stress impairs the response to growth factors and leucine in muscle cells.

          Methods

          Muscle cells were incubated overnight with tunicamycin or thapsigargin to induce ER stress and the activation of the unfolded protein response, mTORC1 activity at baseline and following insulin and amino acids, as well as amino acid transport were determined.

          Results

          ER stress decreased basal phosphorylation of PKB and S6K1 in a dose-dependent manner. In spite of the decrease in basal PKB phosphorylation, insulin (10–50 nM) could still activate both PKB and S6K1. The leucine (2.5–5 mM)-induced phosphorylation of S6K1 on the other hand was repressed by low concentrations of both tunicamycin and thapsigargin. To determine the mechanism underlying this anabolic resistance, several inhibitors of mTORC1 activation were measured. Tunicamycin and thapsigargin did not change the phosphorylation or content of either AMPK or JNK, both increased TRB3 mRNA expression and thapsigargin increased REDD1 mRNA. Tunicamycin and thapsigargin both decreased the basal phosphorylation state of PRAS40. Neither tunicamycin nor thapsigargin prevented phosphorylation of PRAS40 by insulin. However, since PKB is not activated by amino acids, PRAS40 phosphorylation remained low following the addition of leucine. Blocking PKB using a specific inhibitor had the same effect on both PRAS40 and leucine-induced phosphorylation of S6K1.

          Conclusion

          ER stress induces anabolic resistance in muscle cells through a PKB/PRAS40-induced blockade of mTORC1.

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          Most cited references 38

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          Regulation of 4E-BP1 phosphorylation: a novel two-step mechanism.

          The multisubunit eukaryotic translation initiation factor (eIF) 4F recruits 40S ribosomal subunits to the 5' end of mRNA. The eIF4F subunit eIF4E interacts directly with the mRNA 5' cap structure. Assembly of the eIF4F complex is inhibited by a family of repressor polypeptides, the eIF4E-binding proteins (4E-BPs). Binding of the 4E-BPs to eIF4E is regulated by phosphorylation: Hypophosphorylated 4E-BP isoforms interact strongly with eIF4E, whereas hyperphosphorylated isoforms do not. 4E-BP1 is hypophosphorylated in quiescent cells, but is hyperphosphorylated on multiple sites following exposure to a variety of extracellular stimuli. The PI3-kinase/Akt pathway and the kinase FRAP/mTOR signal to 4E-BP1. FRAP/mTOR has been reported to phosphorylate 4E-BP1 directly in vitro. However, it is not known if FRAP/mTOR is responsible for the phosphorylation of all 4E-BP1 sites, nor which sites must be phosphorylated to release 4E-BP1 from eIF4E. To address these questions, a recombinant FRAP/mTOR protein and a FRAP/mTOR immunoprecipitate were utilized in in vitro kinase assays to phosphorylate 4E-BP1. Phosphopeptide mapping of the in vitro-labeled protein yielded two 4E-BP1 phosphopeptides that comigrated with phosphopeptides produced in vivo. Mass spectrometry analysis indicated that these peptides contain phosphorylated Thr-37 and Thr-46. Thr-37 and Thr-46 are efficiently phosphorylated in vitro by FRAP/mTOR when 4E-BP1 is bound to eIF4E. However, phosphorylation at these sites was not associated with a loss of eIF4E binding. Phosphorylated Thr-37 and Thr-46 are detected in all phosphorylated in vivo 4E-BP1 isoforms, including those that interact with eIF4E. Finally, mutational analysis demonstrated that phosphorylation of Thr-37/Thr-46 is required for subsequent phosphorylation of several carboxy-terminal serum-sensitive sites. Taken together, our results suggest that 4E-BP1 phosphorylation by FRAP/mTOR on Thr-37 and Thr-46 is a priming event for subsequent phosphorylation of the carboxy-terminal serum-sensitive sites.
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            Loss of the tuberous sclerosis complex tumor suppressors triggers the unfolded protein response to regulate insulin signaling and apoptosis.

            Mammalian target of rapamycin, mTOR, is a major sensor of nutrient and energy availability in the cell and regulates a variety of cellular processes, including growth, proliferation, and metabolism. Loss of the tuberous sclerosis complex genes (TSC1 or TSC2) leads to constitutive activation of mTOR and downstream signaling elements, resulting in the development of tumors, neurological disorders, and at the cellular level, severe insulin/IGF-1 resistance. Here, we show that loss of TSC1 or TSC2 in cell lines and mouse or human tumors causes endoplasmic reticulum (ER) stress and activates the unfolded protein response (UPR). The resulting ER stress plays a significant role in the mTOR-mediated negative-feedback inhibition of insulin action and increases the vulnerability to apoptosis. These results demonstrate ER stress as a critical component of the pathologies associated with dysregulated mTOR activity and offer the possibility to exploit this mechanism for new therapeutic opportunities.
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              Immobilization induces anabolic resistance in human myofibrillar protein synthesis with low and high dose amino acid infusion.

              We tested the hypothesis that increasing blood amino acid (AA) availability would counter the physical inactivity-induced reduction in muscle protein synthesis. We determined how 14 days of unilateral knee immobilization affected quadriceps myofibrillar protein synthesis (MPS) in young healthy subjects (10 men, 2 women, 21 +/- 1 years; 80.2 +/- 4.0 kg, mean +/- S.E.M.) in the post-absorptive state and after infusing AA (10% Primene) at low or high doses (43 and 261 mg kg(-1) h(-1)). Muscle cross-sectional area (MRI) and peak isometric torque declined in the immobilized leg (-5.0 +/- 1.2% and -25 +/- 3%, respectively, both P 0.6) in the non-immobilized leg. Immobilization induced a 27% decline in the rate of post-absorptive MPS (immobilized, 0.027 +/- 0.003: non-immobilized, 0.037 +/- 0.003% h(-1); P < 0.001). Regardless of dose, AA infusion stimulated a greater rise in MPS in the non-immobilized legs; at 4 h MPS was greater by +54 +/- 12% with low dose and +68 +/- 17% with high dose AA infusion (both P < 0.001). There was some evidence of delayed responsiveness of phosphorylation of Akt to high doses of AA and p70S6k at both doses but no marked differences in that of mTOR, GSK3beta or eEF2. Phosphorylation of focal adhesion kinase (Tyr(576/577)) was reduced (P < 0.05) with immobilization. We observed no change in polyubiquitinated protein content after immobilization. We confirm that 14 days of immobilization reduces MPS in the post-absorptive state and this diminution is reduced but not abolished by increased provision of AA, even at high rates. The immobilization-induced decline in post-absorptive MPS with the 'anabolic resistance' to amino acids can account for much of immobilization-induced muscle atrophy.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2011
                16 June 2011
                : 6
                : 6
                Affiliations
                [1 ]Université catholique de Louvain, Institute of Neuroscience, Research Group in Muscle and Exercise Physiology, Louvain-la-Neuve, Belgium
                [2 ]Research Centre for Exercise and Health, Department of Biomedical Kinesiology, K.U. Leuven, Leuven, Belgium
                [3 ]Université catholique de Louvain, Institut de Recherche Expérimentale et Clinique, Pole of Cardiovascular Research, Woluwe-Saint-Lambert, Belgium
                [4 ]Department of Neurobiology, Physiology and Behaviour, University of California Davis, Davis, California, United States of America
                McMaster University, Canada
                Author notes

                Conceived and designed the experiments: LD LB MF KB. Performed the experiments: LD AP KB. Analyzed the data: LD LB MF KB. Contributed reagents/materials/analysis tools: LD LB MF KB. Wrote the paper: LD MF KB.

                Article
                PONE-D-11-05807
                10.1371/journal.pone.0020993
                3116857
                21698202
                Deldicque et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                Page count
                Pages: 9
                Categories
                Research Article
                Biology
                Molecular Cell Biology
                Cell Growth
                Cellular Stress Responses
                Signal Transduction
                Medicine
                Anatomy and Physiology
                Musculoskeletal System
                Muscle
                Muscle Biochemistry
                Cell Physiology

                Uncategorized

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