Antioxidant Effect Of MTORC1 Signaling Pathway On Sow Mammary Gland And Its Molecular Mechanism Regulated By Methionine

Milk yield and lactation persistence are directly determined by the number of mammary epithelial cells.A well-developed mammary gland in sow is an important guarantee for piglet survival,weaning weight and subsequent growth potential.Late pregnancy and lactation are critical periods for sow mammary gland development,as well as periods of oxidative stress in the body.Oxidative stress not only causes oxidative damage,but also inhibits the mechanistic target of rapamycin complex 1（mTORC1）signaling triggering autophagy and cell death.Intracellular methionine can participate glutathione（GSH）synthesis defensing oxidative stress.Recently,it has been found that S-adenosylmethionine（SAM）,a metabolite of methionine,can directly active mTORC1 signaling.Therefore,we hypothesized that methionine defensed oxidative stress and improved cell viability through activating mTORC1 signaling by SAM.We explored the relationship among mammary oxidative stress,mTORC1 signaling and methionine metabolism using microdialysis and mammary vein cannula during critical period of mammary gland development i.e.late gestation and lactation.Furthermore,we isolated mammary epithelial cell from a female piglet to study the mechanism that methionine defensed oxidative stress and helped cell survival.This thesis is divided into 4 experiments.Experiment 1:Study on the relationship among mammary mTORC1 signaling pathway,metabolism and redox status under physiological conditions.Twelve crossed（Landrace×Yorkshire）primary parity gilts with similar body condition were used in this study.The mammary extracellular fluids and tissues were collected at 60and 90 days of pregnancy,respectively.Compared with 60 days of gestation,the mammary parenchyma at 90 days of gestation had larger lobular,more alveolus,larger size of alveolus.Mammary gland increased（P<0.05）cross-sectional area,wet weight,crude protein contents,and decreased（P<0.05）crude fat at 90 days of pregnancy than at 60 days of pregnancy.In terms of metabolism,Warburg effect,pentose phosphate pathway,nucleotide metabolism and lipolysis were enhanced（P<0.05）at gestation days 90 than 60 in mammary gland.The content of methionine in the breast was strongly correlated with the content of almost all significantly different metabolites（P<0.05,r>0.66）.Compared with 60 days of pregnancy,a higher（P<0.05）level of hydrogen peroxide（H₂O₂）in mammary extracellular fluid and an increase（P<0.05）in the content of glutathione disulfide（GSSG）and 2-hydroxyadenine in mammary gland tissue at 90 days of pregnancy.The protein expression of the downstreams of mTORC1,such as p-S6K1（T389）and p-4E-BP1（T37/46）,were also decreased（P<0.05）at 90 days of pregnancy.Mammary SAM contents at 90 days of pregnancy were 16 times that of at 60 days of pregnancy（P<0.05）,but SAH contents were unaltered（P>0.05）.The above results indicated that mammary gland development in late gestation suffered oxidative stress and had down-regulated the mTORC1 signaling pathway.Under oxidative stress,the consumption of methionine increased,and methionine was more synthesized by the mammary gland into SAM and may be used to maintain a certain degree of activation of the mTORC1 signaling pathway to resist oxidative stress and ensure mammary development.Experiment 2:mTORC1 signaling in mammary gland under oxidative stress caused by feed restriction.Twelve crossed（Landrace×Yorkshire）primary lactating gilts were used in this study and randomly assigned into control and restricted feed allowance（RFA）groups（n=6/group）,respectively.The study lasted 28 days,and the control group was provided 5 kg/d and the RFA group was only provide 3.8 kg/d from lactating days 8 to lactating days 27.At lactating days 28,the RFA group had higher（P<0.05）body weight loss,backfat loss,body fat loss and body protein loss than the control group.In terms of adipose tissue,the RFA group had higher（P<0.05）extracellular glutamate concentrations and insulin contents in adipose tissue than the control group at lactating days 28,otherwise,the RFA group had lower（P<0.05）pyruvate/glucose and lactate/glucose after 90 min and 120 min of ingestion than the control group,respectively.Compared with the RFA group,the control group increased（P<0.05）the gene expression of PKLR,LDHB and VAMP8 in adipose tissue,and decreased the gene expression of ATGL and HSL in adipose tissue.At lactation days 28,the control group had decreased（P<0.05）malondialdehyde concentrations in ear vein than the RFA group,and had higher（P<0.05）glutathione peroxidase activity in ear vein than the RFA group.The RFA group had lower（P<0.05）glutathione peroxidase activity and the contents of taurine and histidine in mammary vein than the control group.Compared with the control group,the RFA group had the downregulation（P<0.05）of p-mTOR（S2448）,p-S6K1（T389）,p-4E-BP1（T37/46）,β-casein and lysosomal v-ATPase activity in mammary gland at lactation days 28.However,there was no difference（P>0.05）of most amino acids,including methionine,and nutrients between two groups,moreover,there was no difference（P>0.05）of milk yield,amino acids contents in milk and piglet performance between two groups.In summary,a 24%feed restriction resulted in decreased insulin sensitivity of fat tissue of lactating sows,which caused catabolism and increased body mobilization.Feed restriction induced oxidative stress in the mammary glands of lactating sows,which down-regulated the lysosomal v-ATPase/mTORC1 signaling pathway and blocked the synthesis ofβ-casein in the mammary glands,and interfered with the differentiation of mammary epithelial cells.Experiment 3:Hydrogen peroxide impacted methionine metabolism and mTORC1signaling in mammary epithelial cells.Mammary epithelial cells were isolated from neonatal female piglets,the study was conducted when more than 94%cells were mammary epithelial cells.After treating mammary epithelial cells with different concentrations of H₂O₂ for several times,the cells were collected and related indexes were determined.The result showed that more than 0.2m M H₂O₂ decreased（P<0.05）cell viability and PCNA protein expression.H₂O₂ increased（P<0.05）malondialdehyde in medium and decreased（P<0.05）intracellular GSH/GSSG and the protein expression of p-ULK1（S757）,p-S6K1（T389）and p-4E-BP1（T37/46）in high-dose and long-term treatment.Moreover,LC3-II protein expressions were increased（P<0.05）in high-dose and long-term H₂O₂ treatment,as was Beclin-1 protein expression.However,H₂O₂ increased（P<0.05）p-S6（S235/236）expression in long-term treatment.Low-concentration and short-term H₂O₂ treatment only increased（P<0.05）SAM synthesis and did not increase（P>0.05）SAH synthesis.Only at high-concentration and long-term H₂O₂ treatment increased（P<0.05）both SAM and SAH synthesis.The above results show that H₂O₂ caused cellular oxidative stress,inhibited the mTORC1 signaling pathway,preferentially synthesized SAM to participate in anti-oxidation,and promoted autophagy.However,the activation of S6K1 depended on the length of H₂O₂ treatment.Experiment 4:Mechanism of methionine to maintain mammary epithelial cells survival through the mTORC1 signaling pathway under oxidative stress.Cells were treated with basal medium containing 0.8 m M H₂O₂,and methionine and its hydroxyl analogs or SAM were added to observe the remission of cell autophagy and activation of the mTORC1 signaling pathway.The methyltransferase inhibitor SGI-1027was used to inhibit the metabolism of SAM to generate SAH,and whether SAM relieves autophagy caused by oxidative stress through the transmethylation pathway.H₂O₂inhibited（P<0.05）cell viability.Methionine and its hydroxyl analogue helped（P<0.05）cell viability under H₂O₂treatment.H₂O₂ increased（P<0.05）the LC3-II expression and inhibited（P<0.05）the relevant protein expression of mTORC1 signaling.Under H₂O₂supplement condition,methionine or SAM increased（P<0.05）the protein expression of the downstream of mTORC1 and decreased（P<0.05）LC3-II protein expression.With the addition of 1μM methyltransferase inhibitor SGI-1027 and 0.8 m M H₂O₂,methionine supplement could still increase（P<0.05）the activation of p-S6K1（T389）.Furthermore,with the addition of 2μM SGI-1027,H₂O₂ could not change（P>0.05）LC3-II and p-S6K1（T389）protein expression.This indicated that SAM synthesized by methionine activated mTORC1 signaling pathway to inhibit autophagy caused by oxidative stress independently of transmethylation.Based on the above test results,we had the following conclusions:oxidative stress caused mammary gland oxidative damage and down-regulated the mammary gland mTORC1 signaling pathway by inhibiting lysosomal v-ATPase activity.Under oxidative stress conditions,mammary epithelial cells preferentially use SAM produced by methionine to directly activate the m OTRC1 signaling pathway,which inhibited the initiation of autophagy and helped cell survival.