Research On The Role And Mechanism Of BAMBI In The Adipogenesis In Mice

BMP and Activin Membrane-Bound Inhibitor(BAMBI)is a transmembrane glycoprotein,and because of its defective intracellular structure,so it is also known as the pseudo-receptor of TGF? signaling.Previous studies have shown that in human adipocytes,the use of si RNA to interfere with the expression of BAMBI could promote the differentiation of human preadipocytes,and can alleviate the inhibition of the classical Wnt/?-catenin signaling pathway and TGF? signaling pathway on adipogenic differentiation of adipocytes effect.This discovery also made us interested in the role of the BAMBI gene in improving the meat quality of meat animals.The results of our laboratory research have also verified that in pig primary preadipocytes,BAMBI could regulate adipogenesis through the classical Wnt/?-catenin signaling pathway.However,so far,there is still insufficient research on the effect of BAMBI gene on adipocyte differentiation,how BAMBI regulates adipogenesis needs further exploration.Therefore,in present study,C57/L6 mice were used as research objects.The Cre-lox P system was used to construct adipose-specific BAMBI knockout mice(BAMBI AKO).The inguinal white adipocytes and brown adipocytes were isolated.And the effects of BAMBI adipogenesis and its mechanism were explored by RT-q PCR,Western blot,transcriptome sequencing,oil red O staining,and immunofluorescence staining at the tissue and cell level.This study also provides a theoretical basis for the improvement of meat quality in livestock.The results obtained are shown below:1.Adipose-specific BAMBI knockout mice were successfully constructed.In present study,the Cre-lox P technology was used toconstruct adipose-specific BAMBI knockout mice(BAMBI AKO).In the inguinal white adipose tissue(i WAT)and epididymal white adipose tissue(e WAT),the m RNA and protein expression level of BAMBI was extremely significantly reduced(P<0.01),indicating that adipose-specific BAMBI knockout mice were successfully constructed;2.BAMBI AKO mice showed an obese phenotype,accompanied by insulin resistance and fatty liver.After 14 weeks of chow diet feeding,there was no significant difference in body weight,tissue weight,and expression level of adipogenic marker genes in the control and BAMBI AKO mice,but after 14 weeks of high-fat diet feeding,the weight of body,liver tissue,i WAT,e WAT,and BAT and adipocyte area of BAMBI AKO mice were increased significantly(P<0.05).The BAMBI AKO mice showed an obese phenotype,and decreased insulin sensitivity.The phosphorylation of Akt(Ser473)signaling pathway in adipose tissue,liver tissue and muscle tissue of BAMBI AKO mice was also significantly reduced,indicating insulin resistance was found in BAMBI AKO mice(P<0.05).In addition,adipose-specific knockout of the BAMBI increased the accumulation of lipids in liver;3.BAMBI knockout promotes adipogenic differentiation of primary preadipocytes in mice.To investigate whether BAMBI could have the same effect on preadipocytes as the effect on adipose tissue,we isolated preadipocyte cells from i WAT and BAT.The results showed adipose-specific knockout of BAMBI caused increased lipid accumulation in white and brown preadipocyte,and the adipogenesis marker genes(PPAR?,C/EBP?,a P2,)were significantly increased in preadipocyte,indicating that BAMBI knockout promoted the adipogenic differentiation of white and brown preadipocytes in mice.Oil Red O and Bodipy staining results also verified the results of real-time PCR and Western blot,suggesting BAMBI has an inhibitory effect on adipogenic differentiation of preadipocytes in mice.4.BAMBI promotes adipogenesis of primary preadipocytes by regulating ROS levels in mice.To further explore the mechanism of BAMBI on adipogenesis,i WAT,e WAT,and BAT of two group mice were sequenced by high-throughput transcriptome sequencing,and 1356,194,and 1300 differentially expressed genes(DEGs)were screened,respectively.The results of RT-q PCR verified the reliability of the sequencing data.In addition,KEGG pathway enrichment analysis shows that DEGs are mainly enriched in the oxidative stress related and adipogenesis related pathway,suggesting that BAMBI may regulate adipogenesis by regulating the level of ROS in mice.In order to investigate whether the hypertrophy of adipose tissue was caused by increased ROS level with high fat diet,we measured the production of ROS in preadipocytes,and the results showed that adipose-specific knockout of BAMBI could increase intracellular ROS levels,and promote mitotic clonal expansion process of preadipocyte at the differentiation stage,and then increase C/EBP? activity,and eventually promote the adipogenesis of primary precadipocyte,and antioxidant N-acetylcysteine could inhibit adipocyte differentiation induced by BAMBI knockout;In summary,in this study,we successfully constructed BAMBI AKO mice,and investigated the role of BAMBI in adipogenesisin vivo and vitro,and concluded that BAMBI may regulate lipogenesis by regulating the level of oxidative stress in mice,and explained its mechanism of action on mouse lipogenesis.The results of this study not only provide a new theoretical basis for the improvement of livestock meat quality and the construction of BAMBI transgenic pig,but also provide new ideas and targets for the treatment of obesity.