A Study On The Mechanism Of Bcl2l13 Promoting The Browning Of White Adipose Tissue

Obesity is one of the most important health problems facing human beings at present,and it has become a global epidemic.According to statistics from the World Health Organization,there are more than 600 million obese adults worldwide.Obesity is an important inducer of various diseases,such as diabetes,insulin resistance,coronary heart disease,stroke,metabolic syndrome,etc.,which seriously threaten human health.The prevention and treatment of obesity is essential to alleviate these health problems.Adipose tissue is an important tissue target for the prevention and treatment of obesity.It is divided into white fat that stores surplus energy and brown fat and beige fat that utilize energy.The production process of beige fat,also known as browning of white fat,has attracted much attention because of its inducibility.In-depth study of the molecular mechanism of browning of white fat will provide a new basis for the mechanism of combating obesity,diabetes,metabolic syndrome and other diseases.As a member of the Bcl-2 family,Bcl2l13 can promote apoptosis,and can also act as a mitophagy receptor,promoting mitochondrial fragmentation and mitophagy.Combining with the previous reports,we found that knockout of the Bcl2l13 gene in mice affects the browning phenotype of white fat,but the detailed mechanism in the process is not yet clear.In this study,Bcl2l13 knockout(Bcl2l13^-/-)mice were used to explore the function and possible mechanism of Bcl2l13 in browning of white fat in vivo.First,we treated wild-type mice with cold stimulation at 4°C orβ3-adrenergic agonist CL316243 to induce the browning of white fat,and found that during this process,the expression of Bcl2l13 in the inguinal adipose tissue of mice increased significantly.Subsequently,we constructed Bcl2l13^-/-mice based on CRISPR/Cas9 in order to study the influence of Bcl2l13 deletion on white fat browning under cold stimulation.We found that during cold stimulation,the body temperature of Bcl2l13^-/-mice was lower than that of the control group.HE staining results showed that the number of beige adipocytes in inguinal adipose tissue of Bcl2l13^-/-mice was significantly less than that of the control group.This result indicated that after ablation of Bcl2l13,the browning process of white fat in mice was significantly inhibited.Real-time quantitative PCR and Western blot were used to detect the expression of brown adipocyte markers.The results of real-time quantitative PCR showed that the m RNA levels of genes involved in the thermogenic program,including Ucp1,Pgc1a,Prdm16,and Cidea,were significantly decreased in Bcl2l13^-/-mice versus control mice.Western blot analysis showed that the expression of UCP1 in inguinal adipose tissue of Bcl2l13^-/-mice was significantly reduced compared with WT mice.To further study the mechanism by which Bcl2l13 regulates the browning process of white fat,we explored the effect of Bcl2l13 deletion on mitochondrial dynamics under cold stimulation.Vascular stromal cells were extracted from Bcl2l13^-/-mice and WT mice,and after browning was induced,the mitochondrial morphology was observed using a confocal electron microscope.The results showed that the mitochondrial morphology of the WT group was dot-shaped,and the mitochondrial morphology of the Bcl2l13^-/-group was elongated.Bcl2l13^-/-mice were cold stimulated with 4℃,and the expression of mitochondrial dynamics related genes in inguinal adipose tissue was detected by real-time quantitative PCR and Western blot.In inguinal adipose tissue,m RNA leves of Drp1,Tfam and Vdac1 were down-regulated in Bcl2l13 knockout mice.Western blot analysis showed that VDAC1 protein expression was largely reduced in inguinal adipose tissue of Bcl2l13^-/-mice.At the same time,a quantitative study of mitochondrial DNA was performed,and the results showed that the amount of mitochondrial DNA was significantly reduced in the inguinal adipose tissue and brown adipose tissue of Bcl2l13^-/-mice compared with the control group.The above results indicate that Bcl2l13 may affect the browning process of white fat by regulating mitochondria dynamics.To further explore the upstream and downstream signaling pathways of Bcl2l13regulating white adipose tissue browning,we first used transcriptome sequencing to study the signaling pathways regulated by Bcl2l13 knockout in adipose tissue under cold stimulation.The results showed that the up-regulated genes were mainly involved in the biological processes such as the regulation of cell activation,the homeostasis of calcium ion and the positive regulation of lipase activity.The down-regulated genes were mainly involved in biological processes such as the response to temperature stimulation,the regulation of norepinephrine secretion,the regulation of lipid metabolic process,the regulation of oxidative phosphorylation,the response to insulin and the production of vascular endothelial growth factor.This result suggests that under cold stimulation,the absence of Bcl2l13 causes the gene expression involved in the biological processes related to white fat browning to be significantly down-regulated,thereby inhibiting the browning of white adipose tissue and thermogenesis.In addition,we also investigated whether the Bcl2l13 interaction protein NINJ2 also affects browning of white adipose tissue.The Biogrid database shows that there is an interaction between Bcl2l13 and NINJ2.In this study,we confirmed that the two were co-localized in mitochondria and there was an interaction through co-localization experiments and co-immunoprecipitation.In addition,Ninj2 knockout mice were treated with cold stimulation,and the expression of UCP1 in inguinal adipose tissue was detected by Western blot.The results showed that the expression of UCP1 in the inguinal adipose tissue of Ninj2 knockout mice was significantly higher than that of WT mice,indicating that ablation of Ninj2 promotes the browning of white adipose tissue.This study is the first to use Bcl2l13 knockout mice and Ninj2 knockout mice to discover the influence of Ninj2-Bcl2l13 regulatory axis on white fat browning in vivo and its preliminary mechanism.This study found that loss of Bcl2l13 inhibited mitochondrial fragmentation,reduced the number of mitochondria,and thus inhibited browning of white fat.Moreover,Ninj2 may affect the browning of white fat in mice through interaction with Bcl2l13.This study preliminarily clarified that Ninj2-Bcl2l13plays an important physiological role in the browning of white adipose tissue.It provides a new mechanism for understanding the dynamic regulation process of mitochondria in browning of white fat,and also provides new ideas for combating obesity and metabolic syndrome.