Effects Of Glucose And Amino Acids On Casein Synthesis In Mammary Gland Cells Of Dairy Goats

Amino acids and energy influence the synthesis of casein.Animal experiments have shown that energy and protein supply has a superimposed effect on casein synthesis.The mammalian target of rapamycin(mTOR)can respond to extracellular stimuli,such as growth factors,energy,and amino acids,and regulate various cellular activities,including protein synthesis.This experiment investigated the regulatory mechanism of glucose and amino acids on casein synthesis in primary mammary gland cells of dairy goats.This study aimed to investigate the effects of glucose alone,amino acids alone,and co-supplementation of glucose and amino acids on casein expression and mTOR pathway-related protein activity.Test 1:Effects of glucose alone on casein synthesis and AMPK-mTOR pathway activity in breast cells.The experiment was based on glucose concentration in the DMEM/F12 culture medium,adding 0,0.25,0.5,0.75,1,1.25,1.5,and 1.75 times glucose,respectively.There were eight treatment groups,and each group was repeated three times.The results showed that when the glucose gradient increased,the relative expression of casein(P=0.006),S6K1(P<0.001),and the phosphorylation ratio of 4EBP1(P<0.001)in breast cells increased first and then decreased.The phosphorylation ratio of AMPK(P<0.001)decreased first and then increased.The results of this experiment indicate that glucose has the effect of promoting casein synthesis by activating the AMPK-mTOR signaling pathway within a specific concentration range,while excessive glucose concentration can inhibit casein synthesis.The optimal concentration for glucose treatment is 17.50 mmol/L.Test 2:Effects of amino acids alone on casein synthesis and mTOR pathway activity in breast cells.The experiment was based on the concentration of amino acids in the DMEM/F12 culture medium,adding 0,0.25,0.5,0.75,1,1.25,1.5,and 1.75 times amino acids,respectively.There were eight treatment groups,and each group was repeated three times.The results showed that when the amino acid gradient increased,the relative expression of casein(P<0.001),S6K1(P<0.001),and the phosphorylation ratio of 4EBP1(P<0.001)in breast cells increased first and then decreased.The results of this experiment indicate that amino acids have the effect of promoting casein synthesis by activating the mTOR signaling pathway within a specific concentration range,while excessive levels can inhibit casein synthesis.The optimal concentration for amino acid treatment is 7.20 mmol/L.Test 3:Effects of co-addition of glucose and amino acids on casein synthesis and AMPKmTOR pathway activity in breast cells.In this experiment,three concentrations of glucose were used:low(8.75 mmol/L),medium(13.125 mmol/L),and high(17.50 mmol/L),and three concentrations of amino acids:low(3.60 mmol/L),medium(5.40 mmol/L),and high(7.20 mmol/L).Orthogonal experiments were conducted in nine treatment groups,and each group was repeated three times.The results showed that compared to the control group,the addition of glucose or amino acids promoted the expression of casein and the phosphorylation ratio of mTOR pathway-related proteins S6K1 and 4EBP1 and inhibited the phosphorylation ratio of AMPK.However,no interaction was observed between the effects of glucose and amino acids,which had an additive effect on the synthesis of casein.Test 4:Transcriptome analysis of casein in breast cells induced by glucose and amino acids.The results showed that the differential genes were mainly enriched in Negative regulation of B cell-mediated immunity compared to the control group,Positive regulation of T cell costimulation,MAPK signaling pathway,Cytoplasmic DNA sensing pathway,AMP-activated protein kinase activity,Triglyceride catabolism process,cAMP metabolic process,AMPK signaling pathway,DNA helicase activity,Rapl signaling pathway,Growth factor receptor binding,Positive regulation of stimulus response and PI3K-AKT signaling pathway.In general,adding glucose or amino acids can promote the synthesis of casein in breast cells.Glucose and amino acids have the effect of regulating milk secretion in dairy cows within a certain concentration range.Excessive concentrations can affect the absorption of breast cells,thereby affecting the synthesis of milk protein.However,no interaction has been observed between the effects of glucose and amino acids,and glucose and amino acids have an additive effect on the synthesis of casein.