One mechanism by which AKT kinase-dependent hypersensitivity to mammalian target of rapamycin (mTOR) inhibitors is controlled is by the differential expression of cyclin D1 and c-MYC. Regulation of post-transcriptional processes has been demonstrated to be crucial in governing expression of these determinants in response to rapamycin. Our previous data suggested that cyclin D1 and c-MYC expression might additionally be coordinately regulated in an AKT-dependent manner at the level of transcription. Under conditions of relatively quiescent AKT activity, treatment of cells with rapamycin resulted in upregulation of cyclin D1 and c-MYC nascent transcription, while in cells containing active AKT, exposure repressed transcription. Promoter analysis identified AKT-dependent rapamycin responsive elements containing AP-1 transactivation sites. Phosphorylated c-JUN binding to these promoters correlated with activation of transcription while JUNB occupancy was associated with promoter repression. Forced overexpression of JunB or a conditionally active JunB-ER allele repressed cyclin D1 and c-MYC promoter activity in quiescent AKT-containing cells following rapamycin exposure. AIP4/Itch-dependent JUNB protein degradation was found to be markedly reduced in active AKT-containing cells compared to cells harboring quiescent AKT. Moreover, silencing AIP4/Itch expression or inhibiting JNK mediated AIP4 activity abrogated the rapamycin-induced effects on cyclin D1 and c-MYC promoter activities. Our findings support a role for the AKT-dependent regulation of AIP4/Itch activity in mediating the differential cyclin D1 and c-MYC transcriptional responses to rapamycin.
The extent to which cellular metabolites are NMR observable is of fundamental importance in the interpretation of in vivo NMR studies. Analysis of ischemic rat liver shows that ATP resonances measured by 31P NMR decrease considerably faster than total tissue ATP measured in extracts. This discrepancy demonstrates that, in liver, ATP is not 100% observable. Furthermore, the data are consistent with the supposition that in situ mitochondrial ATP resonances are not normally observable by in vivo NMR techniques. The specificity of the NMR measurement for cytosolic ATP indicates that 31P NMR can be a valuable tool for the specific measurement of ATP in this compartment.
L-Methionine is the most toxic amino acid if supplied in excess, and the metabolic basis for this toxicity has been extensively studied, with varying conclusions. It is demonstrated here that in vivo 2H NMR spectroscopy provides a useful approach to the study of the hepatic metabolism of methionine in the anesthetized rat. Resonances corresponding to administered L-[methyl-2H3]methionine, and to the transmethylation product sarcosine, are observed during the first 10-min period after an intravenous injection of the labeled methionine, and the time dependence has been followed for a period of 5 h. A third resonance, assigned to the N-trimethyl groups of carnitine, phosphorylcholine, and other metabolites, becomes observable several hours after administration of the deuteriated methionine. In addition, there is a small increase in the intensity of the HDO resonance over the period of the study, which is interpreted to reflect the ultimate oxidation of the labeled sarcosine methyl group via mitochondrial sarcosine dehydrogenase. Additional small 2H resonances assigned to N1-methylhistidine and creatine could be observed in perchloric acid extracts of the livers of rats treated with the deuteriated methionine. Inhibition of the flux through the transmethylation pathway is observed in the rat pretreated with the S-ethyl analogue of methionine, ethionine. These data provide strong support for the importance of glycine transmethylation in the catabolism of excess methionine.
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