Dataset Information

The effects of AICAR and rapamycin on mitochondrial function in immortalized mitochondrial DNA mutator murine embryonic fibroblasts.

ABSTRACT: Mitochondrial DNA mutations accumulate with age and may play a role in stem cell aging as suggested by the premature aging phenotype of mitochondrial DNA polymerase gamma (POLG) exonuclease-deficient mice. Therefore, E1A immortalized murine embryonic fibroblasts (MEFs) from POLG exonuclease-deficient and wild-type (WT) mice were constructed. Surprisingly, when some E1A immortalized MEF lines were cultured in pyruvate-containing media they slowly became addicted to the pyruvate. The POLG exonuclease-deficient MEFs were more sensitive to several mitochondrial inhibitors and showed increased reactive oxygen species (ROS) production under standard conditions. When cultured in pyruvate-containing media, POLG exonuclease-deficient MEFs showed decreased oxygen consumption compared to controls. Increased AMP-activated protein kinase (AMPK) signaling and decreased mammalian target of rapamycin (mTOR) signaling delayed aging and influenced mitochondrial function. Therefore, the effects of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), an AMPK activator, or rapamycin, an mTOR inhibitor, on measures of mitochondrial function were determined. Rapamycin treatment transiently increased respiration only in WT MEFs and, under most conditions, increased ATP levels. Short term AICAR treatment transiently increased ROS production and, under most conditions, decreased ATP levels. Chronic AICAR treatment decreased respiration and ROS production in WT MEFs. These results demonstrate the context-dependent effects of AICAR and rapamycin on mitochondrial function.

SUBMITTER: Delic V

PROVIDER: S-EPMC6262855 | biostudies-other | 2018 Nov

REPOSITORIES: biostudies-other

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The effects of AICAR and rapamycin on mitochondrial function in immortalized mitochondrial DNA mutator murine embryonic fibroblasts.

Delic Vedad V Noble Kenyaria K Zivkovic Sandra S Phan Tam-Anh TA Reynes Christian C Zhang Yumeng Y Phillips Oluwakemi O Claybaker Charles C Ta Yen Y Dinh Vinh B VB Cruz Josean J Prolla Tomas A TA Bradshaw Patrick C PC

Biology open 20181116 11

Mitochondrial DNA mutations accumulate with age and may play a role in stem cell aging as suggested by the premature aging phenotype of mitochondrial DNA polymerase gamma (POLG) exonuclease-deficient mice. Therefore, E1A immortalized murine embryonic fibroblasts (MEFs) from POLG exonuclease-deficient and wild-type (WT) mice were constructed. Surprisingly, when some E1A immortalized MEF lines were cultured in pyruvate-containing media they slowly became addicted to the pyruvate. The POLG exonucle ...[more]

PMID: 30177551

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Project description:Infectious bronchitis virus (IBV), the causative agent of infectious bronchitis, is responsible for major economic losses in the poultry industry worldwide. While IBVs can usually be passaged in primary chicken embryonic fibroblasts (CEFs), most of the wild ones cannot adapt to passaged cell lines. In this study, the wild strain CK/CH/MY/2020 was used to infect primary CEF and immortalize DF-1 CEF cells. Results indicated that IBV was able to cause lesions and pass onto CEF, but not DF-1. Indeed, the virus could enter DF-1 cells and synthesize the associated structural gene but could not assemble into complete viral particles for release. Furthermore, transcriptome sequencing analysis showed significant differences in gene expression between CEF and DF-1 cells after viral infection, although the corresponding antiviral responses could be activated in both cell types. The biggest difference was in terms of the amino acid biosynthesis pathway and the cytokine receptor interaction pathway, which were significantly and specifically activated in CEF. This could actually explain why intact viruses can be assembled but not in DF-1. In addition, SLBP and P2RX7 affect the replication of IBV's structural genes to some extent. Overall, IBV can enter CEF and DF-1 cells, but the complex intracellular cytokine interactions affect the assembly and release of viral particles. The insight will be useful for the study of IBV through in vitro transmission and pathogenesis.ImportanceInfectious bronchitis virus (IBV) is responsible for high morbidity and mortality as well as substantial economic losses worldwide. Transcriptome sequencing of IBV-infected chicken embryonic fibroblast and DF-1 cells revealed that the virus elicits antiviral immunity in cells after viral infection, but IBV cannot activate DF-1 cells to produce sufficient amounts of viral structures to assemble into complete virions, which may be caused by the interactions between cytokines. The study of IBV cellular adaptations is important for vaccine development and investigation of the pathogenesis of IBV.

Project description:BackgroundThe polo-like kinases (Plks) are a group of serine/threonine kinases which have roles in many aspects of cellular function including the regulation of mitotic activity and cellular stress responses. This study focuses on Plk4, the most divergent member of the Plk family, which is necessary for proper cellular proliferation. More specifically, alterations in Plk4 levels cause significantly adverse mitotic defects including abnormal centrosome duplication and aberrant mitotic spindle formation. We sought to clarify the effect of reduced Plk4 levels on the cell by examining transcript profiles of Plk4 wild-type and heterozygous mouse embryonic fibroblasts (MEFs). Subsequently, the levels of several key proteins involved in the DNA damage response were examined.Results143 genes were found to be significantly up-regulated in the heterozygous MEFs compared to their wild-type counterparts, while conversely, 9 genes were down-regulated. Numerous genes with increased transcript levels in heterozygous MEFs were identified to be involved in p53-dependent pathways. Furthermore, examination of the promoter regions of all up- and down-regulated genes revealed that the majority contained putative p53 responsive elements. An analysis of transcript levels in MEFs after exposure to either ionizing or ultraviolet radiation revealed a significant change between wild type and heterozygous MEFS for Plk4 transcript levels upon only UV exposure. Furthermore, changes in protein levels of several important cell check-point and apoptosis regulators were examined, including p53, Chk1, Chk2, Cdc25C and p21. In heterozygous MEFs, p53, p21 and Chk2 protein levels were at significantly higher levels. Furthermore, p53 activity was increased 5 fold in the Plk4 heterozygous MEFs.ConclusionGlobal transcript profiles and levels of key proteins involved in cellular proliferation and DNA damage pathways were examined in wild-type and Plk4 heterozygous MEFs. It was determined that Plk4 haploinsufficiency leads to changes in the levels of RNA accumulation for a number of key cellular genes as well as changes in protein levels for several important cell cycle/DNA damage proteins. We propose a model in which reduced Plk4 levels invoke an increase in p53 levels that leads to the aforementioned changes in global transcription profiles.

Dataset Information

The effects of AICAR and rapamycin on mitochondrial function in immortalized mitochondrial DNA mutator murine embryonic fibroblasts.

Publications

The effects of AICAR and rapamycin on mitochondrial function in immortalized mitochondrial DNA mutator murine embryonic fibroblasts.

Similar Datasets

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