Abstract
BackgroundControl of translation allows for rapid adaptation of the cell to stimuli, rather than the slower transcriptional control. We presume that translational control is an essential process in the control of adipogenesis, especially in the first hours after hormonal stimulation. 3T3-L1 preadipocytes were cultured to confluency and adipogenesis was induced by standard protocols using a hormonal cocktail. Cells were harvested before and 6 hours after hormonal induction. mRNAs attached to ribosomes (polysomal mRNAs) were separated from unbound mRNAs by velocity sedimentation. Pools of polysomal and unbound mRNA fractions were analyzed by microarray analysis. Changes in relative abundance in unbound and polysomal mRNA pools were calculated to detect putative changes in translational activity. Changes of expression levels of selected genes were verified by qPCR and Western blotting.ResultsWe identified 43 genes that shifted towards the polysomal fraction (up-regulated) and 2 genes that shifted towards free mRNA fraction (down-regulated). Interestingly, we found Ghrelin to be down-regulated. Up-regulated genes comprise factors that are nucleic acid binding (eIF4B, HSF1, IRF6, MYC, POLR2a, RPL18, RPL27a, RPL6, RPL7a, RPS18, RPSa, TSC22d3), form part of ribosomes (RPL18, RPL27a, RPL6, RPL7a, RPS18, RPSa), act on the regulation of translation (eIF4B) or transcription (HSF1, IRF6, MYC, TSC22d3). Others act as chaperones (BAG3, HSPA8, HSP90ab1) or in other metabolic or signals transducing processes.ConclusionsWe conclude that a moderate reorganisation of the functionality of the ribosomal machinery and translational activity are very important steps for growth and gene expression control in the initial phase of adipogenesis.
Highlights
Control of translation allows for rapid adaptation of the cell to stimuli, rather than the slower transcriptional control
Adipogenesis was assessed by the analysis of mRNA steady state levels of C/EBPb and PPARg by means of Quantitative Real Time-PCR (q-PCR) in total RNA at time points day 0, 1 and 9
PPARg was up-regulated at day 9, no significant change of mRNA steady state levels were detected at day 1 (Additional file 2)
Summary
Control of translation allows for rapid adaptation of the cell to stimuli, rather than the slower transcriptional control. We presume that translational control is an essential process in the control of adipogenesis, especially in the first hours after hormonal stimulation. 3T3-L1 preadipocytes were cultured to confluency and adipogenesis was induced by standard protocols using a hormonal cocktail. Changes in relative abundance in unbound and polysomal mRNA pools were calculated to detect putative changes in translational activity. Many factors have already been identified, mainly by RNA analysis, which regulate cell fate and control the transformation of preadipocytes into mature adipocytes. This process confers massive changes in the structure of the cell, which leads to storage of fat and the formation of fat vacuoles. Until recently, regulating mechanisms of the earliest step in the differentiation process are poorly understood in comparison to later stages of adipogenesis
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