Effect Of Adra1a On The Differentiation Of Mice White Adipocyte

Obesity is a disease caused by the accumulation of excess adipose tissue,which is a predisposing factor of many clinical diseases.The expansion of adipose tissue during obesity occurs through adipocyte hypertrophy and adipocyte hyperplasia,which involves recruitment,proliferation and differentiation of adipocyte precursors.Adra1 a gene encodes multipathway transmembrane proteins,which play a role in the positive regulation of protein phosphorylation,the regulation of smooth muscle contraction,and the regulation of arterial blood pressure.In our previous studies,we found that Adra1 a can promote the browning of white adipose tissue,but its role and mechanism in the differentiation of white adipose cells are still unclear.In this study,mouse white adipocytes were used as the research object to explore the effect of Adra1 a on the adipogenic differentiation of white adipocytes and its mechanism through in vitro adipogenesis induction and Adra1 a gene overexpression technology.The results are as follows:1.Isolation,culture and identification of white adipocytesIsolate mouse white adipocytes by collagenase digestion and culture them in DMEM medium containing 10% FBS.The cell morphology is typically fibroblast-like.After oil red O staining,many single vesicles can be observed in mature adipocytes.Lipid droplets prove that the isolated white adipocytes obtained in this study are typical white adipocytes.2.Expression of Adra1 a during adipogenic differentiation of white adipocytesThe white adipocytes were differentiated into adipocytes,collect white adipocytes on day 2,4,6 and 8 of differentiation,and detect the expression changes of Adra1 a during adipogenic differentiation by RT-q PCR and Western Blot.It was found that the expression of Adra1 a decreased with the extension of differentiation time.3.Transfection efficiency of Adra1 a overexpression vector and its effect on cell viabilityThe Adra1 a overexpression vector was constructed,and the Adra1 a overexpression vector was transfected into white adipocytes by liposome transfection.The transfection efficiency was tested and it was found that the overexpression vector could increase the expression of Adra1 a by 24.8 times.CCK8 was used to detect the effect of Adra1 a overexpression on the activity of white adipocytes,and it was found that the Adra1 a overexpression vector had no significant effect on cell activity.4.The effect of Adra1 a overexpression on the adipogenic differentiation of white adipocytesWhite adipocytes were transfected with Adra1 a overexpression vector for adipogenesis induction.The changes in the expression levels of adipogenesis related genes were detected by RT-q PCR and Western Blot.The results showed that the transcriptional and protein levels of PPARγ decreased significantly.Oil red O staining and lipid droplets were used to quantitatively detect the adipogenesis of white adipocytes after Adra1 a overexpression.It was found that Adra1 a overexpression could inhibit the accumulation of lipid droplets during adipogenic differentiation of white adipocytes.5.The effect of Adra1 a on white adipocyte differentiation signaling pathwayAfter transfection of the Adra1 a overexpression vector,the changes in AMPK and ERK/MAPK and their phosphorylation levels after differentiation of white adipocytes were detected.The results showed that AMPK levels did not change much after Adra1 a overexpression,but their phosphorylation levels were significantly increased.The expression level of ERK also remained unchanged,and its phosphorylation level was also significantly increased.It is confirmed that Adra1 a may inhibit the differentiation of white adipocytes by increasing the phosphorylation level of AMPK and ERK pathways.