| Objectives: It has been newly discovered that Tudor-SN exists in endoplasmic reticulum and lipid droplets. O. Fresnedo et al. found that Tudor-SN plays important role in maintain lipid homeostasis. Studies showed that steatosis promotes more Tudor-SN distribute in the low density lipid drops, and it is closely related with the secretion of phospholipids. Besides, our microarray results of MDA-MB-231 stable cell line showed that many lipid metabolism related genes have obvious changes once Tudor-SN expression been inhibited. This remind us Tudor-SN may play a role in adipogenesis. Our experiments in Tudor-SN transgenic mouse also showed that Tudor-SN may influence mouse insulin sensitivity by affecting adipogenesis progress. The main purpose of this topic is to discuss the role of Tudor-SN in adipocyte differentiation and its possible mechanism, and the impact on the metabolism of mouse.Methods: We over expressed or knocked down Tudor-SN in 3T3-L1 cells, induced the cells with DMI treatment, then stained the cells with oil red O to identify the adipogenesis. Then we use WT-MEF and Tudor-SN knockout MEF to confirm the results. We checked the m RNA and protein expression pattern of Tudor-SN and PPARγ during the differentiation by western-blot and Real-time PCR. To find the relations between Tudor-SN and PPARγ, we use Co-IP and GST-pull down and immunofluorescence. Luciferase assay and Ch IP were used to identify the transcriptional regulation of Tudor-SN and the way Tudor-SN affect PPARγ target genes’ transcription. To confirm if Tudor-SN knockout will affect histone modification, we use Co-IP to check the expression changes of HDACs during the differentiation progress. We constructed Tudor-SN transgenic(Tg) mouse and fed them chow food and high fat diet respectively for 12 weeks with WT mouse as a control. Then we measured their body, liver and WAT weights. We confirmed the liver steatosis by use oil red O and HE to stain mouse liver frozen section. Glucometer was used to test the fast glucose, and glycogen test kit was used to measure the liver glycogen content. ITT and GTT wereperformed to test the insulin sensitivity. We collected the liver, WAT and primary hepatocyte samples at different time points after insulin stimulation, detected the phosphorylation level of Akt, which reflected the insulin sensitivity of these tissues.Results: Tudor-SN has a positive correlation with adipogenesis, once Tudor-SN has been knocked out in MEF cells, adipogenesis can be totally inhibited. Tudor-SN and PPARγ have show consistency in m RNA and protein level during adipogenesis. They combine with each other via the SN domain of Tudor-SN, and have colocalization in the cells. Tudor-SN’s transcriptional activity is regulated by CEBP/β and Tudor-SN can combines to PPRE region of PPARγ’s target genes. The deficiency of Tudor-SN can recruit more HDACs, thus reduce the histone acetylation level. Tudor-SN transgenic mice are significantly lighter in body and liver weight, but much heavier in WAT weight compare with WT mice after HFD fed. Tg mice also have significantly lighter liver steatosis and a higher individual insulin sensitivity. Liver and WAT tissue from Tg mice are also more sensitive than WT mice after insulin stimulation.Conclusion: Tudor-SN promotes the process of adipocyte differentiation as a PPARγ transcription co-activating factor, regulates adipogenesis related downstream gene transcription through affecting histone acetylation. Tudor-SN over expression can reduces liver steatosis under HFD conditions, and induces a promotion in insulin sensitivity. |
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