Linifanib Exerts Dual Anti-obesity Effect By Regulating Adipocyte Browning And Formation

Obesity,ranked as the sixth risk factor for human diseases,not only reduces the quality of life,but also is the main culprit for causing chronic diseases such as cardiovascular diseases,type 2 diabetes and cancers.Obesity is mainly caused energy imbalance,i.e.,energy intake is greater than consumption,and the excess energy is stored in adipose tissues in the form of triglycerides.Traditionally,adipocytes are mainly divided into two categories: white adipocytes and brown adipocytes.Currently brown adipocytes were found to consist of two types: classic brown adipocytes and beige adipocytes(also known as brite adipocytes).Brown adipocytes are different from white adipocytes in that the expression of uncoupling protein 1(Ucp1)on mitochondrial inner membrane is very high.Therefore,the main function of brown adipocytes is to generate heat through Ucp1 and to consume energy without generating ATP.The non-thrilling thermogenic function of brown fat cells can maintain a constant body temperature,and can also expend the energy absorbed by the body in the form of heat,thus reducing the probability of obesity.However,the sources of these two brown adipocytes are different.Classic brown adipocytes are homologous to muscle cells,but beige adipocytes exist in white adipose tissue.Previous studies have found that beige adipocytes can be transdifferentiated from white adipocytes under certain conditions,such as cold exposure,knockout of specific genes or stimulation of drugs such as retinoid(RA),fibroblast growth factor 21(FGF21).Therefore,beige adipocytes have received extensive attention,and the research in this area is of great significance for the treatment of metabolic diseases.Based on this,we are interested in looking for new small molecule compounds that can promote browning of white adipocytes.In order to find new small molecular compounds that can promote browning of white adipocytes,we used the Ucp1-2A-luciferase mouse model established in our laboratory.In short,the mouse model has luciferase gene knocked in at the last exon of Ucp1 gene,so that luciferase gene can be co-expressed and translated with Ucp1 gene to achieve true and accurate expression of Ucp1 gene by measuring the activity of luciferase.By isolating inguinal adipose-derived mesenchymal stem cells from Ucp1-2A-luciferase mice and culturing them in vitro to differentiate into mature adipocytes,we have discovered a new small molecular compound,Linifanib,that can promote browning of white adipocytes.Subsequently,we conducted the following three studies in vivo and in vitro.First of all,in order to determine whether Linifanib can promote browning in vivo and in vitro,we used adipose-derived mesenchymal stem cells isolated from the groin of Ucp1-2A-luciferase mice to culture and differentiate into mature adipocytes in vitro,and then added different concentrations of Linifanib for 24 hours to measure luciferase activity.The results show that after Linifanib treatment,the expression of Ucp1 increased in a dose-dependent manner,and then we did qPCR and Western blot experiments to determine the expression of Ucp1 at mRNA level and protein level,and the results were consistent with previous experiments.At the same time,we also detected the expression of other thermogenic genes after Linifanib treatment,and the results showed that the expression of these thermogenic genes was up-regulated.Then,we did oxygen consumption test,glycerol release test and mitochondrial related protein test.The experimental results showed that Linifanib increased the basic respiration and maximum respiration of cells,increased the mitochondrial content and glycerol release in cells.Next,we want to explore whether Linifanib also plays the same role in the body.Using Ucp1-2A-luciferase mice,we gavaged mice with Linifanib,and then determined luciferase activity in classic brown adipose tissue and inguinal white adipose tissue of mice.The experiment shows that Ucp1 expression in classic brown adipose tissue and inguinal white adipose tissue is increased by Linifanib treatment.In order to identify the possible target of Linifanib,we examined the effect of Linifanib on signal pathways of various protein kinases(such as STAT3,P38,ERK and AMPK),which can enhance the expression of Ucp1 from previous reports.The results showed that Linifanib significantly reduced the phosphorylation of STAT3,while the other three signaling pathways were not affected.After that,we further examined whether these signal pathways affect the browning of white adipocytes induced by Linifanib.We also selected other agonists or antagonists of these kinases(STAT3 activator: SD19;MEK inhibitor: AZD6244P;38 inhibitor: SB202190 and AMPK inhibitor: Compound C)were used to detect their effects on browning of white adipocytes induced by Linifanib.According to the experimental results,we found that only STAT3 activator SD19 blocked the action of Linifanib.This finding is consistent with the inhibitory effect of Linifanib on STAT3 phosphorylation.We have confirmed that Linifanib promotes browning of white adipocytes by inhibiting the phosphorylation pathway of STAT3.Reports have shown that STAT3 can regulate adipocyte differentiation.In order to examine whether Linifanib inhibits adipogenesis,we studied the effect of Linifanib on adipocyte differentiation.We used 3T3-L1 cell line and treated it with Linifanib during differentiation,followed by oil red O staining.The results showed that Linifanib can effectively prevent adipocyte differentiation in a dose-dependent manner.After that,the quantitative experiment of triglyceride content further confirmed this conclusion.Subsequently,we detected the expression of some adipogenic factors after Linifanib treatment.The results showed that Linifanib reduced the expression of Pparγ,Cebpα and Ap2.In order to further study the mechanism of Linifanib inhibiting adipogenesis,3T3-L1 adipocytes were treated with Linifanib at different time points.Oil red O staining showed that Linifanib could inhibit adipocyte differentiation only when Linifanib was added to the cells two days before differentiation.qPCR analysis of Pparγ and Ap2 further supported this conclusion.At the same time,we found that the density of 3T3-L1 cells decreased after treatment with Linifanib.Therefore,we hypothesized that Linifanib may affect the expansion of mitotic cells.Quantitative analysis of cell number confirmed that Linifanib inhibited cell expansion.To sum up,Linifanib improves cell metabolism by inhibiting STAT3 phosphorylation,promotes browning of white adipocytes and inhibits adipocyte generation.Therefore,we believe that Linifanib may be a potential drug for the treatment of obesity.