Effects Of Activation Of Brown Fat And Browning Of Mammary Gland On The Mammary Gland Development And The Involved Mechanisms In Mice

As an unique organ of mammals,mammary glands secrete milk,which not only offers adequate nutrition for animal growth and development,but also provides a variety of dairy raw materials for human beings.The good development of mammary gland is the precondition for the normal lactation function.Thus,the regulation of mammary gland development is of great significance to both animal production and human health.The mammary gland development in postnatal mammals includes different stages such as puberty stage,gestation,lactation and involution.The pubertal mammary gland development is mainly manifested as the growth of mammary ducts and the formation of terminal end buds(TEB).The quality of pubertal mammary gland development directly affects the development of mammary gland function in the subsequent stages.Besides the mammary epithelial cells,mammary gland also contains a large number of adipocytes.Adipocytes are abundant in the pubertal mammary glands,and gradually decrease and almost disappear accompanied with the process of pregnancy and lactation,and then reappear after weaning.The above phenomenons suggest that adipocytes play an important regulatory role in the mammary gland development.At present,the changes of adipocyte types during the development of mammary gland and the effects of different types of adipocytes on the mammary gland development remain largely unknown.Therefore,the objective of this paper is to investigate the regulatory eggects of different types of adipocytes on the mammary gland development of mice,and to explore the possible underlying mechanism.To this end,we first examined the changes in the morphology of mammary adipocytes and the differences in UCP1 expression of mammary gland during the different stages of mammary gland development of mice in order to reveal the possible association between the types or characteristics of adipocytes and mammary gland development.On this basis,the co-culture model of different types(white and brown)adipocytes and mouse mammary epithelial cells HC11 was used to investigate the effect of white and brown adipocytes on the proliferation of HC11 and its molecular mechanism.Secondly,at the in vivo level,β-adrenergic receptor agonist CL316243 was injected intraperitoneally to activate brown fat and induce the browning of subcutaneous fat in pubertal mice.The effects of CL316243 on the development of mammary gland were evaluated,and the possible underlying mechanisms whereby BAT activation regulates the mammary gland development were explored by the means of lipidomics.Finally,we investigated the effect of conjugated linoleic acid(CLA)on the regulation of the types or characteristics of adipocytes in mice under the condition of high fat diet(HFD),and assessed the effects of CLA-induced changes of adipocytes types or characteristics on improving the damage of mammary gland development caused by HFD.Meanwhile,the regulation role and molecular mechanism of different CLA isomers on the proliferation of HC11 were explored in cell culture model.The results are as follows:1,Changes in the morphology of adipocytes and differences in the expression of UCP1 in the different stages of mammary gland development in mice.The pubertal mammary gland tissue of mice contains brown adipocytes,while the mammary gland tissue in pregnancy and lactation stages only contains white adipocytes.The number of adipocytes in the mammary gland gradually reduced,and the expression level of UCP1 gene in the mammary gland tissue gradually decreased with the process from puberty to gestation and lactation.2,Effects of different types of adipocytes(white and brown)on proliferation of mouse mammary epithelial cells.Primary white adipocytes and brown adipocytes were isolated from mice aged 8 weeks and 18.5 days of gestation.The different types of adipocytes were co-cultured with the mouse mammary epithelial cells HC11.The results showed that the coculture of white adipocytes and HC11 cells with plating density of 0.25:1,0.5:1,1:1 and 2:1could significantly promote the proliferation of HC11 cells.In contrast,the co-culture of brown adipocytes and HC11 cells with plating density of 0.25:1,0.5:1,1:1 and 2:1significantly inhibited the proliferation of HC11 cells.3,Regulatory effects and mechanism of intraperitoneal injection of CL316243 on mammary gland development in pubertal mice.Intraperitoneal injection of CL316243 significantly increased rectal temperature,activated BAT,and increased the temperature of BAT in mice.Meanwhile,intraperitoneal injection of CL316243 could significantly increase the expression of UCP1 in mammary gland tissue and induce browning of mammary WAT.In addition,intraperitoneal injection of CL316243 significantly reduced the mammary gland weight,the number of mammary gland duct branch,the number of TEB and catheter crosssectional area,and inhibited the expression of PCNA.Lipidomics results showed that intraperitoneal injection of CL316243 significantly increased the content of phosphatidylcholine(PC)in serum.Among them,dioleoylphosphocholine(DOPC)(10-100μM)significantly inhibited the proliferation of mammary gland epithelial cells HC11.4,Effect and mechanism of conjugated linoleic acid(CLA)on mammary gland development in mice fed HFD.In vivo,compared to the HFD group,dietary supplementation of 1.5% CLA significantly reduced the body weight,body weight gain and fat content of pubertal mice,and significantly increased the serum IGF-1 level.In addition,CLA supplementation significantaly increased the branches of mammary gland duct,the TEB number and the cross-sectional area.Furthermore,dietary CLA promoted the expression of PCNA,Cyclin D1 and CD36 and activated Erk1/2 signaling pathway.In vitro,50 μM c9,t11-CLA significantly promoted the proliferation of palmitic acid(PA)-inhibited HC11 cells,and significantly increased the expression of proliferation marker proteins such as PCNA and Cyclin D1.In contrast,t10,c12-CLA had no such effect.In addition,50 μM c9,t11-CLA activated the erk1/2 signaling pathway,and blockage of Erk1/2 pathway could reverse the promoting effect of c9,t11-CLA on the proliferation of PA-inhibited HC11 cells and the changes of expression of proliferation marker proteins.In summary,the number of adipocytes in the mammary gland gradually decreased and the characteristics of BAT gradually disappear during the process from puberty to gestation and lactation.The results of co-culture of adipocytes and HC11 cells showed that white adipocytes promoted HC11 proliferation,while brown adipocytes inhibited HC11 proliferation.Activation of brown fat may inhibit mammary gland development through DOPC;In the case of HFD,CLA promoted HC11 proliferation and mammary gland development by activating fatty acid receptor CD36 and the Erk1/2 signaling pathway.Collectively,this paper explored the possibility of regulating the activation of brown fat by drugs or nutrition under different nutritional conditions,thus affecting mammary gland development,and revealed the regulatory effect and mechanism of different types of adipocytes on mammary gland development.These findings not only provide scientific basises for improving mammary gland development and lactation performance,but also have important reference significances for reducing the risk of breast cancer and improving human health.