Testosterone Regulates Adipocyte Differentiation And Epididymal Fat Accumulation In Mice Through Modulating Macrophage Polarization

With the development of society and living conditions,obesity has become a global health problem.Studies have shown that testosterone as an important androgen in males which is involved in a variety of biological processes.The level of serum testosterone is closely related to lipid metabolism.Low serum testosterone is a major reason for obesity of males.Macrophages are important immune cells in the body,and can be activated by the microenvironment to form phenotypes with different functions,which is called macrophage polarization.The classically activated macrophage M1 is characterized by high expression of pro-inflammatory cytokines,while M2 polarization is related to allergy and immune regulation.M2 polarization is divided into three subtypes.In this study,the typical M2 a polarization is object.Studies have shown that the balance of adipose tissue macrophage polarization phenotype is closely related to obesity,but the mechanism is not clear.Therefore,the purpose of this study is to explore the relationship between testosterone and adipocytes differentiation,which can provide a theoretical basis for the study of androgen and obesity.In this study,in vitro,3T3-L1 cells were cultured with different concentrations of testosterone?0-10-5 mol/L?or M1/M2 a polarization conditioned medium with or without testosterone?10-7 mol/L?during the entire pre-adipocyte differentiation process.The degree of adipocytes differentiation was detected by Oil Red O staining and the relative mRNA expression of Adiponectin,Leptin,Cebp b,Ppar a,Il6,Tnf a by RT-qPCR.RAW264.7 macrophages were treated by LPS?1 m g/mL?or IL-4?20 ng/mL?andtestosterone?0-10-5 mol/L?.The relative mRNA expression of M1 and M2 a marker genes were detected by RT-qPCR.The effect of testosterone on macrophage polarization was detected.Western blot was used to detect the phosphorylation level of S⁴⁷³ of Akt,and the signal pathway of testosterone in macrophages was explored.We found that testosterone did not directly affect the differentiation of 3T3-L1 cells.While its regulation of M1 macrophage polarization inhibits differentiation of pre-adipose cells.Testosterone suppress LPS-induced M1 polarization and enhance IL-4-induced M2 a polarization with concentration dependence.Cellular signaling studies indicated that testosterone regulated macrophage polarization through the Gai protein,rather than via androgen receptors,and phosphorylation of Akt.In vivo,we designed a luteinizing hormone receptor?LHR?peptide and injected it with adjuvant into male mice at 3 weeks of age to induce antibodies blocking LHR function and leading to decreased serum testosterone levels.These findings demonstrated that low serum testosterone concentrations resulted in increased epididymal white adipose tissue and M1 macrophages in mice.Testosterone supplementation?TPI?reversed the effects of low serum testosterone concentrations on epididymal white adipose formation.In summary,we firstly demonstrated that testosterone strengthens IL-4-incuded M2 a polarization and inhibits LPS-induced M1 polarization,through the inhibitory regulative Gai and phosphorylation of Akt,rather than via androgen receptors.Testosterone has no direct effects on adipocyte differentiation,however,it inhibits pre-adipocyte differentiation induced by M1 macrophage medium.Serum testosterone in male micegenerated by LHR peptide injection results in the increase of epididymal white adipose tissue,and testosterone supplementation reverses this effect.