Determine the effects of mTORC1 inhibition by rapamycin on mouse liver transcriptome
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ABSTRACT: Liver from RICTOR knockout mice show normal levels of mTORC1 signaling in response to refeeding. With this experiment we sought to compare the effects of Rictor depletion to the effects of mTORC1 inhibition by rapamycin in liver from mice that were fasted and refed. Mice were either fasted for 24hr (n=5), or fasted for 22hr then treated with either 10mg/kg rapamycin suspended in 0.9% NaCl and 2% ethanol at a concentration of 1mg/ml (547mM), or vehicle only. After an additional 2 hr, one group of mice (0 hr time, n=3) was sacrificed, the liver immediately removed, and flash frozen in liquid nitrogen. The remaining mice were given ad libitum access to food and sacrificed after 3hr (n=3), 6hr (n=3), or 12 hr (n=3). Two other nonfasted groups of mice (n=2) were injected with either vehicle or rapamycin and sacrificed after 24hr. Submitter cannot locate the CEL files.
Project description:Recent work using mouse models has revealed that mTORC2, which unlike mTORC1 is not acutely sensitive to rapamycin, plays a key role in the regulation of organismal physiology. The substrates and pathways regulated by mTORC2 are at present relatively unknown Using a mouse model with a targeted deletion of hepatic RICTOR, we investigated the loss of mTORC2 on the murine liver transcriptome Rictor floxed (RKO) and control mice (n=4 per group) were fasted overnight, refed for 3 hr, then sacrificed. Livers were removed, rinsed in PBS, and flash frozen in liquid nitrogen. RNA was extracted and hybridized to Affymetrix Genechip Mouse Gene 1.0 ST arrays
Project description:Liver from RICTOR knockout mice show normal levels of mTORC1 signaling in response to refeeding. With this experiment we sought to compare the effects of Rictor depletion to the effects of mTORC1 inhibition by rapamycin in liver from mice that were fasted and refed.
Project description:Little is known about the roles of Rictor/mTORC2 in the leukemogenesis of AML. Here, we demonstrated that Rictor is essential for the maintenance of MLL-driven leukemia by preventing LSCs from exhaustion. Rictor depletion led to a reactive activation of mTORC1 signaling by facilitating the assembly of mTORC1. Hyperactivated mTORC1 signaling in turn drove LSCs into cycling, compromised the quiescence of LSCs and eventually exhausted their capacity to generate leukemia. At the same time, loss of Rictor had led to a reactive activation of FoxO3a in leukemia cells, which acts as negative feedback to restrain greater over-reactivation of mTORC1 activity and paradoxically protects leukemia cells from exhaustion. Simultaneous depletion of Rictor and FoxO3a enabled rapid exhaustion of MLL LSCs and a quick eradication of MLL leukemia. As such, our present findings highlighted a pivotal regulatory axis of Rictor-FoxO3a in maintaining quiescence and the stemness of LSCs. To understand the critical molecular events caused by Rictor loss in MLL-AF9-driven leukemia,the K+Gâ mice BM cells were sorted from the 1st BMT of RictorÎ/Î(MA9_R1,MA9_R2,MA9_R3) or control(MA9_C1,MA9_C2,MA9_C3), and subjected to microarray analysis on Affymetrix microarrays.Furthermore, the MLL-NRIP3-driven mice model was chosen for further examination.The K+Gâ mice BM cells were sorted from the 1st BMT of RictorÎ/Î(MN3_R1,MN3_R2,MN3_R3) or control(MN3_C1,MN3_C2,MN3_C3), and subjected to microarray analysis on Affymetrix microarrays.
Project description:Recent work using mouse models has revealed that mTORC2, which unlike mTORC1 is not acutely sensitive to rapamycin, plays a key role in the regulation of organismal physiology. The substrates and pathways regulated by mTORC2 are at present relatively unknown Using a mouse model with a targeted deletion of hepatic RICTOR, we investigated the loss of mTORC2 on the murine liver transcriptome
Project description:Gene expression was analyzed by gene array in liver RNA collected from 11-12 week old male IR floxed, LIRKO, IR/FoxO1 floxed and LIRFKO mice either a) following an overnight 24 hr fast, b) 60 min after dextrose (2 g/kg ip) was administered to overnight fasted mice, or c) 6 hr after fasted mice were allowed to refeed on standard chow.
Project description:we examined if the activation of the anabolic program mediated by the activation of the mTorc1 complex in the fasted state could suppress the robust catabolic programing and enhanced Pparα transcriptional of mice with a liver specific defect in mitochondrial long chain fatty acid oxidation (Cpt2L-/- mice). We found that the activation of mTorc1 in the fasted state was not sufficient to repress Pparα responsive genes or ketogenesis.
Project description:Mice were fasted for 18 hr overnight then sacrificed or treated with 13C-U-glucose (2 g/kg ip) and sacrificed 1 hr later by decapitation and liver was immediately freeze-clamped and stored in liquid N2 and then at -80 C. Wild type (IR and IR/FoxO1 floxed) mice were sacrificed after fasting and 1 hr post-glucose treatment. Liver-specific insulin receptor knockout (LIRKO) and insulin receptor/FoxO1 double knockout (LIRFKO) mice were sacrificed 1 hr post glucose treatment.
http://www.nature.com/ncomms/2015/150512/ncomms8079/full/ncomms8079.html
Project description:Control and Liver Insulin Receptor KO mice (LIRKO) were sacrificed in the non-fasted state. RNA was prepared from liver samples and subjected to expression microarray analysis Each array was hybridized with sample derived from 2-3 mice of the same genotype.
Project description:Analysis of Xbp1s overexpression in liver after 24 hr indution or 48 hr induction in LIXs mouse. As a control, WT mice after 2 hr refeed and 24 hr fast without refeed are used for analysis of postprandial gene expression. This microarray study was used to screen for target genes activated by Xbp1s in liver. Results provide important information for the role of Xbp1s during postprandial. Total RNA obtained from liver tissues from mice on Dox200 chow diet for 24 hr or 48 hr. Mice were fasted for 6 hr before sacrifice for liver.
Project description:Little is known about the roles of Rictor/mTORC2 in the leukemogenesis of AML. Here, we demonstrated that Rictor is essential for the maintenance of MLL-driven leukemia by preventing LSCs from exhaustion. Rictor depletion led to a reactive activation of mTORC1 signaling by facilitating the assembly of mTORC1. Hyperactivated mTORC1 signaling in turn drove LSCs into cycling, compromised the quiescence of LSCs and eventually exhausted their capacity to generate leukemia. At the same time, loss of Rictor had led to a reactive activation of FoxO3a in leukemia cells, which acts as negative feedback to restrain greater over-reactivation of mTORC1 activity and paradoxically protects leukemia cells from exhaustion. Simultaneous depletion of Rictor and FoxO3a enabled rapid exhaustion of MLL LSCs and a quick eradication of MLL leukemia. As such, our present findings highlighted a pivotal regulatory axis of Rictor-FoxO3a in maintaining quiescence and the stemness of LSCs.