The Regulatory Molecular Mechanism Of Palmitic Acid On Milk Fat Synthesis In Bovine Mammary Epithelial Cells

Milk production of dairy cows mainly depends on the ability of milk synthesis and the proliferation of bovine mammary epithelial cells(BMECs),and milk fat is an important indicator to evaluate milk quality.In the field of life sciences,exploring the molecular mechanism of milk fat synthesis is an important scientific issue.Fatty acids(FAs)are not only substances that provide energy and components of biofilms,but also regulate gene express ion through cell membrane receptor signaling pathways and transcription factor activation pathways.Fatty acids can promote milk fat synthesis in mammary gland by stimulating the expression of adipogenic genes.Palmitic acid(PA)is the primary substrate of milk fat.Therefore,it can be speculated that in the process of milk fat synthesis,palmitic acid can be used as both a milk fat precursor and a signal molecule that affects milk fat synthesis.As an exogenous signal,palmitate can activate phosphatidyl inositol 3-kinase(PI3K)to regulate cell anabolism.Previous studies in our laboratory have found that PA is involved in the regulation of milk synthesis in BMECs,but the underlying mechanism needs for further study.G protein-coupled receptor class C group 6 member A(GPRC6A)is a newly discovered G protein-coupled receptor that can bind to a variety of ligands.Some studies have found that GPRC6 A participates in milk synthesis in BMECs,but its specific mechanism needs for further study.We used BMECs cultured by tissue block method as the research material in this study.The expression of keratin 18(CK18)in BMECs was identified by immunofluorescence and Western blotting,which proved that BMECs were purified and grown in vitro in good condition.PA at 0,50,100,150,and 200 ?M were added to BMECs cultured in vitro.BODIPY staining and triglyceride(TG)detection showed that,with the increase of PA concentration,lipid droplets in and TG content secreted by BMECs were gradually increased.Western blotting was used to detect changes in related protein expression.At a PA concentration of 100 ?M,it had the best stimulatory effect on milk fat synthesis and the expression and maturation of sterol regulatory element binding protein 1c(SREBP-1c).With the same trend,p-PKC? protein levels were significantly increased and subsequently decreased.These results reveal that PA promotes milk fat synthesis in a dose-dependent manner,100 ?M PA is the optimal concentration,and excess PA might be cytotoxic.In order to study the regulatory molecular mechanism of PA on the expression and maturation of SREBP-1c,PA and the PI3 K inhibitor LY 294002 were added to inhibit the PI3 K signaling pathway.Western blotting detected that inhibition of PI3 K almost completely blocked the stimulation of SREBP-1c expression by PA.PI3 K inhibition experiments reveal that PI3 K is the key mediator of PA-induced SREBP-1c expression.The addition of 100 ?M of PA also inhibited protein kinase C?(PKC?).Western blotting detected that knockdown of PKC? only partially reduced the stimulation of SREBP-1c expression by PA,but eliminated the stimulation of SREBP-1c maturation by PA and partially reduced the stimulation of PA on PI3 K activation.PKC? inhibition experiments indicated that PKC? and PI3 K might have different signaling transduction mechanisms.Western blotting detected that PA did not affect the expression of GPR120,and GPR120 knockdown did not change the stimulation of SREBP-1c signal by PA.GPRC6 A knockdown significantly reduced the phosphorylation of PI3 K and PKC? and the expression and maturation of SREBP-1c.GPRC6 A inhibition experiments reveal that GPRC6 A is a key mediator of PA stimulation on the PI3K/PKC?-SREBP-1c signaling.WB detected that 100 ?M PA significantly increased the protein level of GPRC6 A in BMECs.Immunofluorescence detection found that the fluorescence signal of GPRC6 A formed a thin and round structure,indicating that GPRC6 A was located on the plasma membrane of BMECs.Immunofluorescence detection revealed that 100 ?M PA significantly increased the cell membrane localization of GPRC6 A.The results demonstrate that PA promotes GPRC6 A expression and plasma membrane localization in a dose-dependent manner,and PA might be a new agonist of GPRC6 A.In summary,PA dose-dependently regulates the expression and plasma localization of GPRC6 A,activation of PKC,and expression and maturation of SREBP-1c.Excess PA might be toxic to cells.PA promotes milk fat synthesis in B MECs through the GPRC6A-PI3K/PKC? signaling pathway.This study provides theoretical and experimental basis for further utilization of PA in milk fat production.