Dataset Information

Metabolomic Analysis of Mouse Embryonic Fibroblast Cells in Response to Autophagy Induced by Acute Starvation.

ABSTRACT: Autophagy-related protein 7 (Atg7) is essential in the formation of the autophagophore and is indispensable for autophagy induction. Autophagy will exist in lower level or even be blocked in cells without Atg7. Even though the possible signaling pathways of Atg7 have been proposed, the metabolomic responses under acute starvation in cells with and without Atg7 have not been elucidated. This study therefore was designed and aimed to reveal the metabolomics of Atg7-dependent autophagy through metabolomic analysis of Atg7^-/- mouse embryonic fibroblast cells (MEFs) and wild-type MEFs along with the starvation time. 30 significantly altered metabolites were identified in response to nutrient stress, which were mainly associated with amino acid, energy, carbohydrate, and lipid metabolism. For the wild-type MEFs, the induction of autophagy protected cell survival with some up-regulated lipids during the first two hours' starvation, while the subsequent apoptosis resulted in the decrease of cell viability after four hours' starvation. For the Atg7^-/- MEFs, apoptosis perhaps led to the deactivation of tricarboxylic acid (TCA) cycle due to the lack of autophagy, which resulted in the immediate drop of cellular viability under starvation. These results contributed to the metabolomic study and provided new insights into the mechanism associated with Atg7-dependent autophagy.

SUBMITTER: Shen S

PROVIDER: S-EPMC5050436 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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Metabolomic Analysis of Mouse Embryonic Fibroblast Cells in Response to Autophagy Induced by Acute Starvation.

Shen Sensen S Weng Rui R Li Linnan L Xu Xinyuan X Bai Yu Y Liu Huwei H

Scientific reports 20161005

Autophagy-related protein 7 (Atg7) is essential in the formation of the autophagophore and is indispensable for autophagy induction. Autophagy will exist in lower level or even be blocked in cells without Atg7. Even though the possible signaling pathways of Atg7 have been proposed, the metabolomic responses under acute starvation in cells with and without Atg7 have not been elucidated. This study therefore was designed and aimed to reveal the metabolomics of Atg7-dependent autophagy through meta ...[more]

PMID: 27703171

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Project description:BIRC5/Survivin is known as a dual cellular functions protein that directly regulates both apoptosis and mitosis in embryonic cells during embryogenesis and in cancer cells during tumorigenesis and tumor metastasis. However, BIRC5 has seldom been demonstrated as a direct macroautophagy/autophagy regulator in cells. ATG7 expression and ATG12-ATG5-ATG16L1 complex formation are crucial for the phagophore elongation during autophagy in mammalian cells. In this study, we observed that the protein expression levels of BIRC5 and ATG7 were inversely correlated, whereas the expression levels of BIRC5 and SQSTM1/p62 were positively correlated in normal breast tissues and tumor tissues. Mechanistically, we found that BIRC5 negatively modulates the protein stability of ATG7 and physically binds to the ATG12-ATG5 conjugate, preventing the formation of the ATG12-ATG5-ATG16L1 protein complex in human cancer (MDA-MB-231, MCF7, and A549) and mouse embryonic fibroblast (MEF) cells. We also observed a concurrent physical dissociation between BIRC5 and ATG12-ATG5 (but not CASP3/caspase-3) and upregulation of autophagy in MDA-MB-231 and A549 cells under serum-deprived conditions. Importantly, despite the fact that upregulation of autophagy is widely thought to promote DNA repair in cells under genotoxic stress, we found that BIRC5 maintains DNA integrity through autophagy negative-modulations in both human cancer and MEF cells under non-stressed conditions. In conclusion, our study reveals a novel role of BIRC5 in cancer cells as a direct regulator of autophagy. BIRC5 may act as a "bridging molecule", which regulates the interplay between mitosis, apoptosis, and autophagy in embryonic and cancer cells.AbbreviationsACTA1: actin; ATG: autophagy related; BIRC: baculoviral inhibitor of apoptosis repeat-containing; BAF: bafilomycin A1; CQ: chloroquine; CASP3: caspase 3; HSPB1/Hsp27: heat shock protein family B (small) member 1/heat shock protein 27; IAPs: inhibitors of apoptosis proteins; IP: immunoprecipitation; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; PLA: proximity ligation assay; SQSTM1/p62: sequestosome 1; siRNA: small interfering RNA.

Project description:BackgroundAutophagy is a vesicular trafficking process responsible for the degradation of long-lived, misfolded or abnormal proteins, as well as damaged or surplus organelles. Abnormalities of the autophagic activity may result in the accumulation of protein aggregates, organelle dysfunction, and autophagy disorders were associated with various diseases. Hence, mechanisms of autophagy regulation are under exploration.MethodsOver-expression of hsa-miR-376a1 (shortly MIR376A) was performed to evaluate its effects on autophagy. Autophagy-related targets of the miRNA were predicted using Microcosm Targets and MIRanda bioinformatics tools and experimentally validated. Endogenous miRNA was blocked using antagomirs and the effects on target expression and autophagy were analyzed. Luciferase tests were performed to confirm that 3' UTR sequences in target genes were functional. Differential expression of MIR376A and the related MIR376B was compared using TaqMan quantitative PCR.ResultsHere, we demonstrated that, a microRNA (miRNA) from the DLK1/GTL2 gene cluster, MIR376A, played an important role in autophagy regulation. We showed that, amino acid and serum starvation-induced autophagy was blocked by MIR376A overexpression in MCF-7 and Huh7 cells. MIR376A shared the same seed sequence and had overlapping targets with MIR376B, and similarly blocked the expression of key autophagy proteins ATG4C and BECN1 (Beclin 1). Indeed, 3' UTR sequences in the mRNA of these autophagy proteins were responsive to MIR376A in luciferase assays. Antagomir tests showed that, endogenous MIR376A was participating to the control of ATG4C and BECN1 transcript and protein levels. Moreover, blockage of endogenous MIR376A accelerated starvation-induced autophagic activity. Interestingly, MIR376A and MIR376B levels were increased with different kinetics in response to starvation stress and tissue-specific level differences were also observed, pointing out to an overlapping but miRNA-specific biological role.ConclusionsOur findings underline the importance of miRNAs encoded by the DLK1/GTL2 gene cluster in stress-response control mechanisms, and introduce MIR376A as a new regulator of autophagy.

Dataset Information

Metabolomic Analysis of Mouse Embryonic Fibroblast Cells in Response to Autophagy Induced by Acute Starvation.

Publications

Metabolomic Analysis of Mouse Embryonic Fibroblast Cells in Response to Autophagy Induced by Acute Starvation.

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