The Role Of Upstream Rheb And FKBP38in Adipocyte Differentiation In MTOR Signaling Pathway

Rheb (Ras homolog enriched in brain) is a homolog of small G-protein Ras, andfunctions as a small GTPase. There are two types of Rheb in human body, GDP-Rheb andGTP-Rheb, and the later is active. Rheb regulates many progresses via mTOR such as cellsurvival, growth, proliferation, regeneration, energy metabolism, oxidative stress, and soon. In recently, it is believed that hyperirritable mTOR can inhibit lipolysis, promoteadipogenesis and triglyceride storage. But suppression activation of mTOR can decreaselargely the body fat and resist the obesity of high fat diet induced. Because of upstream ofmTOR, we want to clarify whether Rheb plays a significant role in adipogenesis. Weconstructed the recombinant plasmid of pCAG-Insulator-Rheb, which was injected into B6mouse embryo to gain the transgenic mouse of Rheb which is over-expression in the wholebody. And we extracted the mouse embryonic fibroblast cells at pregnancy day13.5, whichwere induced differentiation into adipocytes. Then we detected the amount of triglyceridein MEFs and the expression of adipocyte specific transcript factors PPARγ and C/EBPα atdifferentiation day12. In our experiment, compared to the Rheb negative MEFs, Rhebpositive MEFs manifest high triglyceride, high expression of adipocyte specific transcriptfactors PPARγ and C/EBPα, and the difference of Oil Red O stain. In conclusion,over-expression of Rheb can promote adipocyte differentiation. And the effect may be theresults of over-activation mTOR, which could be explained by the phosphorylation levelsof S6K and S6, being downstream of mTOR. Maybe this will propose a new theory for ourfurther understanding the mechanism of adipocyte differentiation, and an opportunity forpreventing and treating obesity.In body, FKBP38is believed to be an endogenous inhibitor of mTOR, because it caninhibit the function of mTOR by directly binding, similarly to Rapamycin-FKBP12complex. At the same time, FKBP38can bind to GTP-Rheb, releasing mTOR fromFKBP38. But there is still no consensus about the regulation of FKBP38on mTOR, evenmore, these results are at opposite poles. Amino acids, as a classic regulator of mTOR, donot play a role in the regulation of FKBP38on mTOR. Because of the particular site ofFKBP38in mTOR signaling pathway, downstream of Rheb and upstream of mTOR,understanding the regulation mechanism is still very important for us. We also constructedthe recombinant plasmid of pCAG-Insulator-FKBP38, which was injected into B6mouse embryo to gain the transgenic mouse of FKBP38which is over-expression in the wholebody. And extracted MEFs for inducing differentiate into adipocytes. Contrast to Rheb,FKBP38can inhibit adipogenesis, but the mechanism is still need clarified.Objective:(1) To observe the role of Rheb in adipocyte differentiation in mTOR signalingpathway.(2) To observe the role of FKBP38in adipocyte differentiation in mTORsignaling pathway.Methods:(1)Construction recombinant plasmid of pCAG-Insulator-Rheb andpCAG-Insulator-FKBP38, which were injected into mouse embryo for producingtransgenic mice.(2) Identified positive or negative of transgenic mice by PCR, and theexpression amount of Rheb and FKBP38protein by Western blot.(3) Extracted the mouseembryonic fibroblast cells(MEFs) of the transgenic mice on day13.5, and induced them todifferentiate into adipocytes.(4) Stained the MEFs by Oil Red O on day12, and measuredthe amount of triglyceride in the MEFs by the special TG kit, and detected the expressionof special transcription factor PPARγand C/EBPαby the method of Real Time-PCR.Results:(1) Over-expression Rheb can promote adipogenesis in MEFs, and the amount oftriglyceride is incresaed largely(P<0.05), Oil Red O Stain is obviously different. And theexpression of adipocyte specific transcript factor PPARγå'ŒC/EBPα is decreasedenomously（P<0.05）。(2) Over-expression FKBP38can inhibit adipogenesis in MEFs, and the amountof triglyceride is decresaed largely(P<0.05), Oil Red O Stain is obviously different. Andthe expression of adipocyte specific transcript factor PPARγå'ŒC/EBPα is decreasedenomously（P<0.05）。Conclusion:(1)Rheb can promote MEFs differentiate into adipocytes.(2)FKBP38can inhibit MEFs differentiate into adipocytes.