OLA1 Maintains Mouse Fat Mass Stable Through Inhibiting GSK-3?'s Activity

Background:OLA1(Obg-like ATPase 1)5 which belongs to Obg-HfIX superfamily and YchF subfamily,is a translation-factor-related(TRAFAC)GTPase.OLA1's function includes not only hydrolyzing ATP and GTP but also binding ribosomes to regulate protein translation.It plays an important role in signal transduction,intracellular transport,cellular stress response and embryogenesis.Based on our former results,Olal null mice exhibited high peirinatal lethality with survival rate of 20%.These mice had small body size when they grew up.Here we will further analyze the mechanism of this phenotype,and due to the high rate of perinatal lethality,we need to introduce a much more sustainable model to explore OLA1's function,such as tissue-specific knockout mice model.Methods and contents:We use gene trapping and Cre-loxp methods to generate global knockout mice(Ola1-/-)and adipose tissue specific knockout mice(AKO).Then observe the phenotypes of these transgenic mice fed with normal diet or high fat diet.We analyse the metabolism related parameters,like body weight,fat mass,energy consumption and behavioral changes.Generation of primary mouse fibroblasts cell lines(MEFs)which are isolated from E14.5 Ola1-/-embryos and Ola1+/+ embryos.Screen OLA1 interacting proteins with reverse phase protein array(RPPA)from O/a1-/-and Olal+/+ MEF cells.Based on the phenotypes and results of RPPA,we focus on GSK-3?,a potential OLA1 interacting protein.Then we dissect the mechanisms of the interaction between OLA1 and GSK-3? preliminarily.Results:Our O1a1 null mice presented the phenotypes of development delay,growth retardation and high perinatal lethality,indicating the key role of OLA1 in development,which we published before.Here we analyze the adult mice's phenotypes.We found that adult Ola1-/-mice still have small body size.The ratios of organ/femur length are no differences with regard to most of organs of Ola1-/-mice except white adipose tissue.Thus we established a tissue specific Ola1 knockout mice model,adipose tissue specific knockout mice(AKO).Under normal diet or high fat diet,the fat content including white adipose tissue and brown adipose tissue of AKO mice was similar to the Ola1 wild type mice.Then,we found that increased energy consumption,which was the result of increased locomotion activity and decreased food intake,resulted in decreased white adipose tissue content.Meanwhile,a lot of differential expressed proteins were screened by reverse phase protein array.Among of them,GSK-3? was overexpressed accompanied with hyperactivity.Using western blotting and immunohistochemistry,we verified that downregulation of OLA1 in cells,embryos and brain tissues can induce high level of GSK-3P and subsequent upregulation of its protein kinase activity.With qRT-PCR,polysome profiling and CHX chase assay,we attributed elevated GSK-3?protein level in Ola1-/-MEF cells to increased protein stability.We also found that OLA1 inhibited GSK-3?'s protein kinase activity via directly interacting with each other.Taking all things together,we concluded that OLA1 impacted mice fat mass directly through interacting with GSK-3?.Conclusion:In a word,out data demonstrate first time that OLA1,a translational GTPase,is an essential regulator of whole body fat mass through inhibiting GSK-3?'s activity.It influences GSK-3?'s half life and inhibits its protein kinase activity via direct interaction.GSK-3?'s protein level and activity increase in Ola1-/-cells and tissues.Furthermore,increased GSK-3?'s activity in mice brain results in a series of behavioral changes including high locomotor activity and low food intake.These findings suggest that OLA1 is a potential GSK-3?'s inhibitor,and may play an important role in maintaining mouse fat mass stable and whole body energy metabolism.