| Milk fat is one of important nutrient of milk,and the content of milk fat is one of standard to measure milk quality.Milk fat synthesis is regulated by several pathways,the studies suggest that the mechanistic target of rapamycin(mTOR)plays a central role in synthetic process of milk fat.mTOR is a serine/threonine protein kinase,belongs to the phosphatidylinositol 3-kinase-related kinase(PIKK)family of protein kinases.mTOR as the core component,combine with other proteins form two complexs:mTOR complex 1(mTORC1)and mTOR complex 2(mTORC2).Two complexs regulate different cellular processes:mTORC1 regulates such as cell proliferation,metabolism andprotein synthesis.mTORC2 regulates such as actin cytoskeleton,metabolism,and cell migration.Comparing to mTORC2,mTORC 1 is studied much more for regulating milk fat synthesis,and there is no report about comparing the effect in milk fat synthesis between mTORC1 and mTORC2.In the present study,we selected the bovine mammary epithelial cells which were separated by our lab as the research subjects.First using AZD8055(ATP-competitive mTOR inhibitor)to inhibit the activity of mTORC1 and mTORC2.Then we deteced the activity of mTORC1 and mTORC2 signaling using Western blot.Futhermore,the content of triglyceride(TG),palmitic acid(PA),docosahexaenoic Acid(DHA)in the cell supernatant by ELISA;nextly,on the one hand we used rapamycin to inhibit mTORC1 activity,and targeted silence the component of mTORC1 raptor to inhibit mTORC1 activity;on the other hand we targeted silence the component of mTORC2 rictor to inhibit mTORG2 activity.Then we detected mTORC 1 and mTORC2 signaling activity by Western blot assay and detected the content of triglyceride(TG),palmitic acid(PA),docosahexaenoic Acid(DHA)in the cell supernatant by ELISA.Futher more we measured intracellular fatty acid content in the rapamycin treated and the rictor silencing groups by mass spectrum(MS);Finally,the mTORCl and mTORC2 function on milk fat synthesis process were compared using we got datas when both and respective inhibited mTORC 1 and mTORC2 activity.The results showed:1)MTT assay suggested cell proliferation were inhibited by 100 nM AZD8055 treated for 12 h(p>0.05),100 nM AZD8055 treated for 24 h(p>0.05),200 nM AZD8055 treated for 12 h(p>0.05)and 200 nM AZD8055 treated for 24 h(p<0.05);the mTORC1 and mTORC2 acivatity were all inhibited after the cells were treated by AZD8055.The results of ELISA suggested the content of TG,PA and DHA in cell supernatant in control groups were 19.26±0.85 mmol/L,28.89±1.59 ng/ml and 258.75 ±22.1 pg/ml;the content of TG and PA and DHA were 16.07±2.05 mmol/L.22.68±3.2 ng/ml and 188.63±14.57 pg/ml respectivly in cell supernatant in the 100 nM AZD8055 treated for 24 h groups;the content of TG,PA and DHA in cell supernatant were 14.74±2.61 mmol/L?19.54±1.53 ng/ml and 183.82±26.94 pg/ml in the 200 nM AZD8055 treated for 12 h.Compare with control,the content of TG,PA and DHA is significant reducedin treated groups(p<0.05);2)the results of Western blot suggested that the mTORC1 activity were inhibited after BMECs treated by rapamycin;the results of ELISA suggested that the content of TG,PA and DHA in cell supernatant in control group were 5.42±0.08 mmol/L?2.74±0.13 ng/ml and 403.3± 11.17 pg/ml;the total amount of intracellular 31 fat acids was 3070.54±105.07 mg/kg in control group;the content of TG,PA and DHA in cell supernatant in 100 nM rapamycin treated for 8 h groups were 4.53±0.13 mmol/L?2.74±0.13 ng/ml and 297.82±20.8 pg/ml.The content of TG,PA and DHA in the treated groups were reduced significantly(p<0.05)compared with control,total amount of intracellular 31 fat acids were also decreased(p>0.05);3)the results of Western blot suggested the mTORC1 activation were inhibited in the cells of the raptor gene silencing.The results of ELISA suggested the content of TG,PA and DHA in cell supernatant were 8.54±0.8 mmol/L?15.37±1.85 ng/ml and 527.99±32.67 pg/ml in control groups;the content of TG,PA and DHA in cell supernatant were 6.56±0.42 mmol/L?11.07±1.46 ng/ml and 331.84±48.02 pg/ml after raptor gene were slienced.The content of TG,PA and DHA is reduced significantiy in the treated groups compared with control(p<0.05);4)the results of Western blot suggested the mTORC2 activation were inhibited in the cells of the rictor gene silencing,.The results of ELISA suggested the content of TG,PA and DHA in cell supernatant were 3.4±0.43 mmol/L?8.91±1.14 ng/ml and 109.47±7.46 pg/ml in the control group,and the total amount of intracellular 31 fat acids was 3070.54±105.07 mg/kg in the control group;while the content of TG,PA and DHA in cell supernatant after rictor gene slienceing were 2.49±0.24 mmol/L?6.93±0.14 ng/ml and 55.74±6.01 pg/ml,the total amount of intracellular 31 fat acids was 3028.66±120.05 mg/kg.The content of TG,PA and DHA were reduced significantly in the treated groups compared with control(p<0.05),and the total amount of intracellular 31 fat acids also decrease(p>0.05);5)We compared the function of mTORC1 and mTORC2 on TG,PA and DHA systhesis process through three measures.On the one hand,according to the inhibition ratio of TG,PA and DHA to compared the function of mTORC 1 and mTORC2 on TG,PA and DHA systhesis process for TG,PA and DHA,and we founded that the function of mTORC2 were most significant than mTORC1;On the other hand,we compared the function of mTORC1 and mTORC2 in TG,PA and DHA systhesis process according to the inhibition ratio TG,PA and DHA and the singaling activation of mTORC1 and mTORC2.The results showed the function of mTORC2 were most significant than mTORC1 for TG and DHA,and the function of mTORC1 were most significant than mTORC2 for PA.To sum up in conclusions,mTORC1 and mTORC2 both played the key roles for regulation on the synthetic process of TG,PA and DHA in BMECs.Through different measure to compare mTORC1 and mTORC2 effect intensity in TG,PA and DHA synthesis process,we found the effect of mTORC2 may stronger than mTORCl.Because mTORC1 and mTORC1 have cross talking mechanism,the coordination mechanism of mTORC1 and mTORC2 in milk fat synthesis process need to be further studied. |
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