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1.
Magn Reson Med ; 85(1): 560-569, 2021 01.
Article in English | MEDLINE | ID: mdl-32905631

ABSTRACT

PURPOSE: The pharmacokinetics of 3-methoxycarbonyl- and 3-hydroxymethyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl radicals (MCP and HMP, respectively), magnetic resonance probes to assess the brain redox status, were examined in healthy mouse brains. METHODS: The time course of the concentration of the radical form of the probe in the brain was examined by signal enhancements on T1 -weighted MR image after an intravenous injection. The distribution of the total probe (sum of radical and reduced forms) was investigated using brain homogenates. RESULTS: MCP distributed to the brain more than HMP. MCP exhibited biphasic decay with fast and slow components, whereas HMP exhibited monophasic decay with a similar rate constant to the slow component of MCP. Similar profiles were observed in various regions of the brain. The total probe for MCP exhibited monophasic decay at a similar rate constant to the slow component of the radical form; however, the initial content of the total probe was similar to its radical form. For HMP, decay of the total probe coincided with that of the radical form. CONCLUSION: The decay of MCP needs to consider the reduction of the probe in and its elimination from the brain, while the decay of HMP may mainly result from its elimination from the brain.


Subject(s)
Brain , Magnetic Resonance Imaging , Animals , Brain/diagnostic imaging , Cyclic N-Oxides , Electron Spin Resonance Spectroscopy , Injections, Intravenous , Mice , Oxidation-Reduction
2.
J Biol Chem ; 292(44): 18052-18061, 2017 11 03.
Article in English | MEDLINE | ID: mdl-28808055

ABSTRACT

The mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of protein synthesis and potential target for modifying cellular metabolism in various conditions, including cancer and aging. mTORC1 activity is tightly regulated by the availability of extracellular amino acids, and previous studies have revealed that amino acids in the extracellular fluid are transported to the lysosomal lumen. There, amino acids induce recruitment of cytoplasmic mTORC1 to the lysosome by the Rag GTPases, followed by mTORC1 activation by the small GTPase Ras homolog enriched in brain (Rheb). However, how the extracellular amino acids reach the lysosomal lumen and activate mTORC1 remains unclear. Here, we show that amino acid uptake by dynamin-dependent endocytosis plays a critical role in mTORC1 activation. We found that mTORC1 is inactivated when endocytosis is inhibited by overexpression of a dominant-negative form of dynamin 2 or by pharmacological inhibition of dynamin or clathrin. Consistently, the recruitment of mTORC1 to the lysosome was suppressed by the dynamin inhibition. The activity and lysosomal recruitment of mTORC1 were rescued by increasing intracellular amino acids via cycloheximide exposure or by Rag overexpression, indicating that amino acid deprivation is the main cause of mTORC1 inactivation via the dynamin inhibition. We further show that endocytosis inhibition does not induce autophagy even though mTORC1 inactivation is known to strongly induce autophagy. These findings open new perspectives for the use of endocytosis inhibitors as potential agents that can effectively inhibit nutrient utilization and shut down the upstream signals that activate mTORC1.


Subject(s)
Amino Acids/metabolism , Dynamin II/metabolism , Endocytosis , Mechanistic Target of Rapamycin Complex 1/metabolism , Monomeric GTP-Binding Proteins/metabolism , Ras Homolog Enriched in Brain Protein/metabolism , Amino Acid Substitution , Autophagy/drug effects , Biomarkers/metabolism , Clathrin/antagonists & inhibitors , Clathrin/metabolism , Cytoplasm/drug effects , Cytoplasm/metabolism , Dynamin II/antagonists & inhibitors , Dynamin II/genetics , Endocytosis/drug effects , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , HEK293 Cells , Humans , Lysosomes/drug effects , Lysosomes/metabolism , Mechanistic Target of Rapamycin Complex 1/agonists , Mechanistic Target of Rapamycin Complex 1/antagonists & inhibitors , Mechanistic Target of Rapamycin Complex 1/genetics , Membrane Transport Modulators/pharmacology , Microscopy, Fluorescence , Monomeric GTP-Binding Proteins/genetics , Mutation , Protein Transport/drug effects , Ras Homolog Enriched in Brain Protein/genetics , Recombinant Fusion Proteins/metabolism
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