Effects And Molecular Mechanisms Of Fatty Acids On The Mammary Gland Development In Pubertal Mice

Mammary gland organs have the function of secreting milk for the growth,development,and health of mammalian neonates.Therefore,studies on the mammary gland development and its regulation have great significance in improving animal lactation performance and the development of their offspring.Mammary gland development after birth occurs through several distinct stages,including puberty,pregnancy,lactation,and involution.It has been demonstrated that the poor development of mammary gland at puberty has adverse effects on the mammary development of the subsequent stages（especially lactation）.Thus,ensuring the optimal mammary gland development at pubertal stage contributes to the better mammary gland structure and function in subsequent stages.Pubertal mammary gland development is influenced by hormones such as growth hormone（GH）and estrogen,as well as insulin-like growth factor-1（IGF-1）.And nutrition can also affect the mammary gland development through regulating the hormones levels.It has been reported that high-fat diet（HFD）is able to suppress mammary gland development of pubertal mice.However,the underlying mechanisms involved in this process remains clear.In addition,there are few reports about the effects of specific fatty acid on the mammary gland development of pubertal mice.The aim the present study is to elucidate the effect of fatty acids on the mammary gland development in pubertal mice and to explore the possible underlying mechanisms.Firstly,we investigated the effect of HFD on mammary gland development in pubertal mice and the involved molecular mechanism.Secondly,we explored the effects of different fatty acids（butyric acid,lauric acid,palmitic acid,stearic acid,oleic acid and linoleic acid）on the proliferation of mice mammary gland cell HC11,and selected lauric acid（C12:0,LA）,stearic acid（C18:0,SA）and oleic acid（C18:1,OA）in our subsequent studies.On the one hand,in vitro,we determined the effects of lauric acid,stearic acid and oleic acid on the proliferation phenotype,proliferation-related gene expression and proliferation-related pathways in the mice mammary epithelial cell line HC11.On the other hand,in vivo,we assessed the effects of dietary supplementation of lauric acid,stearic acid and oleic acid on the serum hormone levels,ductal branching,TEB number,proliferation related protein and signaling pathway of mammary gland in pubertal mice.The results are as follows:1,HFD suppressed mammary gland development of pubertal mice partly through stimulation of inflammatory signals and inhibition of proliferative signals:Although HFD did not affect on the energy intake of pubertal mice,it significantly increased the mass of mammary gland,the ratio of mammary gland mass to body mass,and markedly decreased the number of mammary gland ducts and TEB number.In addition,the expression of IGF-1,p-Akt and p-Erk in mammary gland tissues was significantly inhibited by HFD,wherea the expression of TLR4 and p-JNK was significantly enhanced.2,LA stimulated mammary gland development of pubertal mice through activation of GPR84 and PI3K/Akt signaling pathway:（1）100μM LA significantly promoted the proliferation of HC11 cells,increased the mRNA/protein expression of proliferation related genes such as Cyclin D1/3 and PCNA,and decreased the protein expression of p21.（2）100μM LA activated PI3K/Akt signaling pathway in dependent on the activation of G protein coupled receptor 84（GPR84）.In addition,the stimulative effects of LA on HC11proliferation and the changes of proliferation related genes expression were reversed by PI3K inhibition or GPR84 knockdown with its interfering RNA.（3）Dietary supplementation of 1%LA had no effect on body weight,average weekly feed intake and fourth pair of mammary gland index.However,LA significantly increased the serum levels of estradiol（E2）and IGF-1,the ductal branching and TEB number of mammry gland.In addition,LA significantly enhanced the expression of GPR84 and Cyclin D1,and activated PI3K/Akt signal pathway.3,OA stimulated mammary gland development of pubertal mice through activation of CD36 and PI3K/Akt signaling pathway:（1）100μM OA significantly promoted the proliferation of HC11 cells,increased the mRNA/protein expression of proliferation related genes such as Cyclin D1/3 and PCNA,and decreased the protein expression of p21.（2）100μM OA activated the fatty acid receptor CD36,increased intracellular Ca²⁺concentration,and then activated CaMKII and PI3K/Akt signaling pathway.In addition,the stimulative effects of LA on HC11 proliferation were reversed by CD36 knockdown with its interfering RNA,chelation of intracellular Ca²⁺,or inhibition of CaMKII and PI3K PI3K.Furthermore,the changes of proliferation related genes expression were abolished by CD36 knockdown or PI3K inhibitor.（3）Dietary supplementation of 2%OA had no effect on body weight,average weekly feed intake and fourth pair of mammary gland index.However,OA significantly increased the ductal branching and TEB number of mammry gland in pubertal mice.4,SA suppressed mammary gland development of pubertal mice through activation of GPR120 and inhibition of PI3K/Akt signaling pathway:（1）40μM SA inhibited the proliferation of HC11 cells,decreased the mRNA/protein expression of proliferation related genes such as Cyclin D1/3 and PCNA,and increased the protein expression of p21.（2）40μM SA enhanced the expression of GPR120 and its downstream target protein PLC-β,while inhibited PI3K/Akt signaling pathway.In addition,the inhibitory effects of SA on HC11proliferation and the changes of proliferation related genes expression were reversed by GPR120 knockdown with its interfering RNA.（3）Dietary supplementation of 2%SA had no effect on body weight,average weekly feed intake and fourth pair of mammary gland index.However,SA significantly decreased the ductal branching and TEB number of mammry gland.In addition,LA significantly enhanced the expression of GPR120,and inhibited the activation of PI3K/Akt signal pathway as well as the expression of Cyclin D1.In summary,HFD suppressed mammary gland development of pubertal mice partly by stimulating of inflammatory signals and inhibiting of proliferative signals.LA,a middle-chain saturated fatty acid,stimulated mammary gland development of pubertal mice and proliferation of HC11 cells through activation of GPR84 and PI3K/Akt signaling pathway.OA,a long-chain monounsaturated fatty acid,stimulated mammary gland development of pubertal mice and proliferation of HC11 cells through activation of CD36-Ca²⁺-CaMKII PI3K/Akt signaling pathway.SA,a long-chain saturated fatty acid,inhibited mammary gland development of pubertal mice and proliferation of HC11 cells through activation of GPR120-PLC-βand inhibition of PI3K/Akt signaling pathway.