Unknown,Transcriptomics,Genomics,Proteomics

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Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type and PPARbeta-null mice during liver regeneration

ABSTRACT: Purpose: The current study tests the hypothesis that peroxisome proliferator-activated receptor Î² (PPARÎ²) has a role in liver regeneration due to its effect in regulating energy homeostasis and cell proliferation. Methods: Wild-type (WT) and PPARÎ²-null mice (KO) male mice (3-5 month, C57BL/6) were kept in steel microisolator cages at 22Â°C with a 14-hr/10-hr light/dark cycle. Food and water were provided ad libitum throughout the entire feeding period. Standard PH was performed. All the animal experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals under protocols approved by the University of California Davis Animal Care and Use Committee.Mouse liver RNA was prepared using TRIzol (Invitrogen, Carlsbad, CA). RNA concentration and integrity were determined by the Agilent 2100 Bioanalyzer using a RNA Nano Bioanalysis Chip. RNA-sequencing library preparation and sequencing was carried out by the Genome Sequencing Facility at University of Kansas Medical Center (Kansas City, KS). cDNA libraries were prepared with 2 Âµg of total RNA using the TruSeq RNA Sample Preparation Kit (Illumina). The libraries were clustered and sequenced on an Illumina HiSeq 2000 instrument with 100 bp paired end reads. Results: Differential gene expression profiling revealed the inhibition of expression in genes and pathways that are involved in metabolism and proliferation in regenerating KO livers. Specifically, PPARÎ² deficiency affected the activation of Akt and the expression of E2fs. Pathways that control glycolysis, FA synthesis as well as cell proliferation were de-regulated in regenerating PPARÎ² KO livers. Conclusions: The data suggest a role for PPARÎ² in regulating liver regeneration that is mediated at least in part through Akt and E2f-regulated pathways. mRNA profiles of 3-month old wild type (WT) and PPARbeta mice were generated by deep sequencing, one simple, using Illumina HiSeq 2000 instrument with 100 bp paired end reads

ORGANISM(S): Mus musculus

SUBMITTER: HUIXIN LIU

PROVIDER: E-GEOD-47062 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Consumption of a protein containing meal by a fasted animal promotes protein accretion in skeletal muscle, in part through leucine stimulation of protein synthesis and indirectly through repression of protein degradation mediated by its metabolite, Î±-ketoisocaproate. Mice lacking the mitochondrial branched-chain aminotransferase (BCATm/Bcat2), that interconverts leucine and Î±-ketoisocaproate, exhibit elevated protein turnover. Here, the transcriptomes of gastrocnemius muscle from BCATm knockout (KO) and wildtype mice were compared using Next Generation RNA-Sequencing (RNA-Seq) to identify potential adaptations associated with their persistently altered nutrient signaling. Statistically significant changes in the abundance of 1486/~39,010 genes were identified. Bioinformatics analysis of the RNA-Seq data indicated that pathways involved in protein synthesis (eIF2, mTOR, eIF4 and p70S6K pathways including 40S and 60S ribosomal proteins), protein breakdown (e.g., ubiquitin mediated), and muscle degeneration (apoptosis, atrophy, myopathy and cell death) were up-regulated. Also in agreement with our previous observations, the abundance of mRNAs associated with reduced body size, glycemia, plasma insulin, and lipid signaling pathways were observed in BCATm KO mice. Consistently, genes encoding anaerobic and/or oxidative metabolism of carbohydrate, fatty acids and BCAAs were modestly but systematically reduced. Although there was no indication that muscle fiber type was different between KO and wildtype mice, a difference in the abundance of mRNAs associated with a muscular dystrophy phenotype was observed, consistent with the published exercise intolerance of these mice. The results suggest transcriptional adaptations occur in BCATm KO mice that along with altered nutrient signaling may contribute to their previously reported protein turnover, metabolic and exercise phenotypes. Comparison of wildtype and BCATm KO gastrocnemius biological replicates

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Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type and PPARbeta-null mice during liver regeneration

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