| In the pig production,maintaining proper body condition is an important guarantee for improving the reproductive performance and extending the longevity of the sow.On the one hand,obesity-induced insulin resistance during pregnancy can reduce the feed intake,affect the growth of piglets,consume the body fat reserves of lactating sows,and prolong the weaning-to-estrus interval of sows;On the other hand,obesity during pregnancy promotes lipid ectopic deposition to the placenta,thereby increasing placental lipotoxicity,causing placental dysfunction,inhibiting placental angiogenesis and fetal growth.Therefore,controlling the backfat of pregnant sows and improving perinatal insulin sensitivity may be one of the key measures to alleviate the decline in reproductive efficiency caused by over-fertilization of sows.Silent information regulator1?SIRT1?is a NAD+-dependent protein deacetylase that plays an important role in regulating fat development,energy metabolism and inflammation.Previous studies have found that inflammation caused by obesity inhibits SIRT1 expression in adipose tissue and aggravates insulin resistance.However,the molecular mechanism is still not fully understood,and the role of SIRT1 in placental development and placental angiogenesis remains to be further explored.Therefore,we firstly used SIRT1 knockout mice to study the function of SIRT1 in adipose tissue inflammation and insulin resistance caused by obesity,and to explore the molecular mechanism of obesity regulating SIRT1 expression in adipose tissue;Subsequently,we investigated the effect of gestational obesity on the expression of SIRT1 in placenta,and the function of SIRT1 in placental angiogenesis and fetal development.Lastly,we added n-3 polyunsaturated fatty acids through the gestational diet to explore whether it can express the placenta SIRT1 to alleviate placental inflammation and oxidative stress induced by high-fat diets,and promotes placental angiogenesis and fetal development.Part ? The role of SIRT1 in obesity-induced inflammation and insulin resistance.In this study,for high fat diet?HFD?-induced obesity studies,mice with different genotypes were systematically assigned to four groups?n=6–8 mice/group?.:?I?SIRT1+/+mice placed on a low fat diet?LFD??10%kcal fat,LFD?;?II?SIRT1+/+mice fed a high-fat diet?60%kcal fat,HFD?;?III?SIRT1+/-mice fed a HFD;?IV?SIRT1-/-mice fed a HFD.The diet for each group was started at 6 weeks of age and sustained for12weeks.After 12 weeks,glucose tolerance and insulin resistance tests were performed and then slaughtered.The main results are as follows:1.Body composition analysis showed that the rats in the HFD group had significant obesity?P<0.05?,and the SIRT1-/-knockout mice had the highest BMI index?P<0.05?,SIRT1+/-mice white adipose tissue?WAT?and brown adipose tissue?BAT?were the highest weight?P<0.05?.Compared with the SIRT1+/-heterozygous mouse group,the SIRT1-/-knockout group had a 20%reduction in WAT content and a 58%reduction in BAT.There was no significant difference in liver weight between the HFD groups.2.Detection of adipose tissue macrophage surface antigen in epididymis showed that the expression of F4/80 and CD11 in SIRT1+/-and SIRT1-/-mice was significantly higher than that in wild-type mice?P<0.05?,but the difference between SIRT1+/-and SIRT1-/-is not significant.Plasma levels of inflammatory cytokines TNF?,IL-1?and IL-6 were significantly increased in SIRT1-/-knockout mice?P<0.05?.3.Glucose tolerance results showed that the glucose tolerance of SIRT1+/-and SIRT1-/-mice was significantly lower than that of wild-type mice?P<0.05?;Insulin tolerance results showed no significant difference between the HFD groups.These results indicate that SIRT1 deficiency intensifies adipose tissue inflammation,and insulin-resistance in HFD-fed mice.Part ? Molecular mechanism of down-regulating SIRT1 during obesity induced inflammation.This study attempted to explore the role of specific miRNAs in affecting adipose tissue inflammation and insulin sensitivity by regulating SIRT1 expression in HFD-induced mouse obesity models and TNF?-induced 3T3-L1 insulin resistance models.The main results are as follows:1.By bioinformatics prediction combined with published sequencing results,we screened 19 candidate inflammation-related miRNAs targeting SIRT1.The 19 miRNAs were co-transfected into BHK cells with the SIRT1 3?-UTR dual luciferase reporter vector,and miR-221,miR-377,miR-145a and miR-103 were found to have the most significant inhibition of luciferase activity?P<0.01?.2.Overexpression of miR-221,miR-377,miR-145a and miR-103 in mature 3T3-L1adipocytes revealed that miR-377 had the most significant inhibitory effect on SIRT1protein?P<0.01?.The double fluorescence results showed that miR-377 significantly inhibited the SIRT1 3'UTR activity?P<0.01?,overexpression of miR-377 had no effect on the luciferase activity of the mutant reporter.3.The miR-377 expression in mature 3T3-L1 cells was significantly increased after exposure to TNF??P<0.05?.Incontrast,SIRT1 mRNA and protein levels were markedly reduced.Similar changes in miR-377 expression were observed in the epididymis adipose tissue of HFD-fed mice.4.It was further found that overexpression of miR-377 in mature 3T3-L1 adipocytes significantly up-regulated the inflammatory signaling pathway and inhibited the insulin signaling pathway;on the contrary,inhibiting miR-377 significantly attenuated TNF?-induced inflammation and insulin resistance.5.The anti-inflammatory and anti-insulin-resistant effects of inhibiting miR-377were significantly inhibited by the use of SIRT1-specific inhibitors EX527.These results indicate that miR-377 functions as a novel inhibitor of SIRT1 in obesity-induced inflammation and insulin-resistance.miR-377 upregulation results in post-transcriptional SIRT1 silencing,which contributes to adipose tissue inflammation and insulin-resistancePart ? Effect of obesity during pregnancy on the expression of placental SIRT1 and lipotoxicity.In this study,20 suitable backfat thickness?18-19mm?and high backfat thickness?>23mm?large white sows were selected,randomly select one placenta per sow to study the effect of obesity on the expression of placental SIRT1 and lipotoxicity.And the SIRT1+/-male and female mice were mated and sacrificed at 18.5 days of gestation to study the effect of SIRT1 deletion on placental angiogenesis and fetal development.The main results are as follows:1.Obesity during pregnancy significantly inhibited the expression of vascular endothelial cell surface antigen CD31 and vascular endothelial growth factor VEGFA?P<0.05?,and the expression of SIRT1 was also significantly decreased?P<0.05?.2.Extraction of placental DNA from sows revealed that obesity during pregnancy had no significantly effect on the content of Cytochrome B in the placenta mitochondrial,but the expression of the key transcription factor PGC1?,which regulates mitochondrial biosynthesis,was significantly down-regulated?P<0.05?.3.Extraction of placental total fat found that the total fat content of placenta in obese sows was significantly increased?P<0.05?,and the expression of placental antioxidant enzymes NRF2 and HO1 was significantly inhibited,placental inflammatory genes TNF?,IL-1?,TLR4,TLR2 and MCP1 were notably activated,indicated that obesity during pregnancy caused placental lipid ectopic deposition and increased lipotoxicity.4.By using SIRT knockout mice,we found that SIRT1 knockout had no effect on placental weight and placental efficiency,but notably reduced fetal weight?P<0.05?,while some SIRT1 knocked out fetal head deformities.Detection of placental angiogenesis-related genes revealed that SIRT1 knockdown significantly reduced the expression of vascular endothelial cell marker gene CD31?P<0.05?and had no significant effect on HIF1?signaling pathway.These results suggests that inhibition of SIRT1 expression by obesity during pregnancy may be an important cause of elevated placental lipid toxicity and limited placental angiogenesis.Part ? Effect of EPA Supplementation on Placental Angiogenesis in SIRT1 Knockout Mice.In this study,50 SIRT1+/-heterozygous male and female mice were used for mating.Pregnant mice were randomly assigned to two groups?n=22/each group?.Group 1received a 60%kcal HFD?including 30.1%lard?.Group 2 received a 60%kcal EPA diet with 25.6%lard and 4.4%EPA-ethyl ester?EPA-EE?.For intraperitoneal glucose tolerance tests?IPGTTs?and intraperitoneal insulin tolerance tests?IPITTs?,mice were fasted for 6h prior to injection glucose at E16.5 or insulin at E17.5?n=6 mice/group?,respectively.And fluorescently labeled fatty acid transport experiments were also performed at E17.5?n=5mice/group?.All mice were sacrificed at 18.5 days of gestation.The purpose of this study was to explore whether the addition of n-3polyunsaturated fatty acids to the diet during pregnancy could promote placental angiogenesis and fetal growth by activating SIRT1 expression and reducing placental lipotoxicity.The main results are as follows:1.EPA treatment significantly increased maternal glucose tolerance?P<0.01?,decreased maternal serum IL-6 and TNF?levels?P<0.01?,and markedly increased the content of saturated fatty acids and n-3 polyunsaturated fatty acids in maternal plasma,reduces n-6 polyunsaturated fatty acids content.2.Detection of placenta and fetal development found that there was no significant effect on placental weight and placental morphology by EPA treatment,but notably increased placental efficiency?P<0.01?and fetal weight?P<0.01?.Immunohistochemistry results showed that EPA significantly promoted the expression of placental angiogenesis marker gene CD31?P<0.01?and inhibited the hypoxia-inducible factor signaling pathway?P<0.01?.In contrast,SIRT1 knockout significantly reduced placental weight?P<0.05?and fetal weight?P<0.01?,markedly reduced placental maze area?P<0.05?and inhibited placental angiogenesis?P<0.01?,there was no significant interaction between dietary factors and genotypes.3.Detection of placental inflammation found that dietary EPA significantly activated the placental inflammatory signaling pathway,up-regulated the expression of a series of inflammatory genes,and promoted the inflammatory core transcription factor NF?B into the nucleus?P<0.01?;SIRT1 knockout did not increase placental inflammation,and EPA treatment had no effect on placental SIRT1 protein levels,there was no significant interaction between dietary factors and genotypes.4.Detection of placental and fetal oxidative stress found that dietary EPA increased total placental antioxidant capacity?T-AOC??P<0.05?and total superoxide dismutase activity?T-SOD??P<0.05?.However,the lipid peroxidation product malondialdehyde?MDA?content was also significantly increased?P<0.05?;SIRT1 knockout did not increase the oxidative stress of the placenta and fetus,and the placental T-SOD activity was significantly enhanced?P<0.05?.5.Detection of placenta and fetal fatty acid composition found that EPA treatment increased the proportion of placenta and fetal saturated fatty acids and n-3polyunsaturated fatty acids;SIRT1 knockdown had little effect on placental fatty acid composition,but significantly reduced the proportion of fetal n-3 polyunsaturated fatty acids.6.Detection of placental lipid metabolism found that EPA treatment significantly reduced placental triglyceride?TG??P<0.01?and total cholesterol?T-CHO?content?P<0.05?,increased the lipolysis genes expression and inhibited lipogenesis genes;SIRT1knockout had no significant effect on placental lipid content;neither EPA treatment nor SIRT1 knockout affected the placenta transport of fluorescently labeled C16 fatty acids.7.Detecting the mitochondrial function of placenta found that EPA notably up-regulated the PGC1?protein level?P<0.05?,while SIRT1 knockdown significantly inhibited the expression of PGC1??P<0.01?;Both EPA treatment and SIRT1 knockout had no effect on mitochondrial DNA content.Electron microscopy results found that EPA had little effect on placental mitochondria morphology,while SIRT1 knockout mitochondria showed increased cyst and vacuolization.These results indicate that SIRT1 knockout can inhibit placental angiogenesis and fetal development,while,maternal EPA feeding promotes placental angiogenesis through a SIRT1 independent inflammatory pathway.In summary:1.HFD-induced obesity can inhibit the translation of SIRT1 by up-regulating the expression of miR-377 in adipose,and resulting in aggravation of adipose inflammation and systemic insulin resistance.2.Obesity during pregnancy can inhibit the placental SIRT1/PGC1?expression and increase placental lipotoxicity,thereby inhibiting placental angiogenesis.3.Under the condition of HFD duiring pregnancy,partial replacement of saturated fatty acids with EPA promotes placental angiogenesis and fetal growth by activating the SIRT1-independent inflammatory pathway. |
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