| Background:Gestational diabetes mellitus(GDM)is defined as first recognition or glucose intolerance with onset during pregnancy.Besides,GDM affects approximately 17%of pregnancies worldwide as one of the most common pregnancy complications.Particularly,in China,approximately 2.9 million pregnant women are suffering with GDM,as the diversified Chinese dietary pattern.GDM have short-term or long-term adverse consequences for both pregnant women and offspring.However,GDM cannot be diagnosed until the middle or early third trimester with serious complications.Therefore,studies on modifiable risk factors in early pregnancy will be more conducive to the early prevention of GDM.In addition,GDM is a complex condition that includes high risks of insulin resistance and chronic low-grade inflammatory responses.In view of the fact that physiological hyperlipidemia and high-fat diet during pregnancy lead to increased LPS level in the serum,which contribute to the elevated expression of inflammatory factors in peripheral insulin target organs.Abnormal activation of inflammatory factors and the production of the ROS results in the high expression of CEBPβ-HDAC1 complex and decreases intracellular NAD~+expression,subsequently inhibiting SIRT1promoter activity and ultimately contributing to decreased insulin sensitivity.This study explores the synergistic effects of hyperlipidemia and high-fat diet patterns on abnormal intestinal gram-negative bacteria proliferation,increased LPS production,and up-regulated expression of inflammatory factors in peripheral insulin target organs,reduced intracellular NAD~+synthesis,increased CEBPβ-HDAC1 complex synthesis,and inhibition of SIRT1 promoter activity.Eventually,the decreased insulin sensitivity and the occurrence of insulin resistance in pregnant women is involved in the pathogenesis of GDM.Objective:The pathogenesis of GDM is associated with the adverse health outcomes of pregnant women and fetus,and the insulin target organ is important for the occurrence of insulin resistance.This study aimed to investigate the effects of LPS-mediated HDAC1 and SIRT1 related protein and insulin resistance on insulin target organs,so as to reveal the function and significance of inflammatory factors and insulin function in GDM.Importantly,our study demonstrates a new viewpoint and scientific basis for investigating the mechanism of GDM.Methods:(1)Serum samples of normal pregnant women and GDM patients were collected.The concentrations of LPS,TC and TG in serum were detected by ELISA Kit.Gas Chromatography-flame ionization detector(GC-FID)was used for quantitative fatty acid detection.The adipose and skeletal muscle tissues of normal pregnant women and GDM patients were collected,and the inflammatory factors TNF-α,NF-κB,histone deacetylase(HDAC1),CAAT/enhancer binding proteinβ(CEBPβ),NMNAT-1,SIRT1 and insulin signaling pathway PI3K/AKT were measured by Western blotting.(2)HL7702 Cells(L02 cells)was induced with low concentration of LPS to investigate the effects and mechanism of LPS on liver inflammatory environment and insulin resistance.q RT-PCR was used to detect L02 inflammatory cytokines IL-1,IL-6 and TLR-4 after LPS inducing.The protein expression of TNF-α,NF-κB,HDAC1 and SIRT1 related protein and IRS-1/PI3K/AKT pathway were detected by Western blotting.ELISA Kit was performed to detect the levels of cellular glucose and insulin,NAD~+;Reactive oxygen species(ROS)kit was used to detect ROS levels.The expression of NMNAT-1 was measured by immunofluorescence.(3)TLR-4 inhibitor TAK-242 were added in L02cells with to observe whether TAK-242 could rescue the expression of LPS-induced downstream pathways.q RT-PCR was performed to investigate L02 inflammatory factors IL-1,IL-6 and TLR-4 after TAK-242treatment.The protein expression of TNF-α,NF-κB,HDAC1 and SIRT1related protein and IRS-1/PI3K/AKT pathway were detected by Western blotting.(4)The lentivirus was used to knockdown HDAC1 in L02 cells and the expression of TAK-242 was observed.q RT-PCR was used to detect inflammatory cytokines IL-1,IL-6 and TLR-4 after HDAC1 knockdown in L02 cells.The protein expression of TNF-α,NF-κB,HDAC1and SIRT1related protein and IRS-1/PI3K/AKT pathway were measured by Western blotting.(5)C57BL/6J mice were fed low fat diet(LFD)to establish the control group,and fed high fat diet(HFD)to establish the GDM group model.The GDM model was weighed,and oral glucose tolerance test(OGTT)and insulin level were detected.At 15.5-17.5 days of gestation in the successfully constructed GDM group,3mg/kg TAK-242 was injected daily into the tail vein of the GDM mice.The serum,liver,adipose tissue and skeletal muscle of mice were collected.The protein levels of TNF-α,NF-κB,HDAC1 and SIRT1 related protein and PI3K/AKT pathway were investigated in adipose tissue and skeletal muscle of mice by the Western blotting.ELISA kit was used to detect the levels of insulin,LPS,TC and TG.Reactive oxygen species(ROS)kit was used to detect ROS levels.Hematoxylin-eosin staining(HE)was used to observe the liver and adipose tissue structure of mice in the three groups.The expression of SIRT1 in adipose tissue was observed by using immunohistochemical.The structures of liver and skeletal muscle of three group of mice were observed by transmission electron microscope(TEM).Results:(1)LPS concentration in serum of GDM patients was higher than that of normal pregnant women.The expression of HDAC1 in adipose tissue and skeletal muscle of GDM patients was higher than that of normal pregnant women,and the expression of NMNAT-1 and SIRT1 was down-regulated.The phosphorylated protein expression of PI3K/AKT pathway in adipose tissue and skeletal muscle of GDM patients was lower than normal pregnant women.(2)LPS increased the levels of inflammatory factors,decreased the expression of insulin signaling pathway and increased glucose concentration in L02 cells 24h later.The protein expression of HDAC1 and CEBPβin LPS-induced L02 cells was higher than that in normal cells,accompanied by down-regulation of NMNAT-1 and SIRT1protein expression.The TAK-242 restored the protein expression level of HDAC1 and SIRT1 related protein.The protein expression of insulin signaling pathway IRS-1/PI3K/AKT in L02 cells after LPS added was lower than that in control cells,and the TAK-242 treatment could rescue the protein expression of insulin signaling pathway IRS-1/PI3K/AKT.After HDAC1 knockdown,CEBPβprotein expression was down-regulated while NMNAT-1 and SIRT1 protein expression were up-regulated.In addition,the phosphorylated protein expression of IRS-1/PI3K/AKT of insulin signaling pathway could be restored after knockdown HDAC1.(3)In HFD-induced GDM mice model,the level of inflammatory factors elevated,while the expression of insulin signaling pathway decreased.Besides,the insulin concentration increased in insulin target organs(liver,adipose tissue and skeletal muscle).The protein expression of HDAC1 and CEBPβin insulin target organs of GDM mice was higher than that of normal pregnant mice,and the protein expression of NMNAT-1 and SIRT1 was down-regulated.The function of TAK-242 rescued the protein expression level of HDAC1 and SIRT1 related protein.The protein expression of PI3K/AKT in insulin signaling pathway in insulin target organs(liver,adipose tissue and skeletal muscle)of mice in the GDM mice was decreased than in the control mice and TAK-242 could rescued the phosphorylated protein expression of PI3K/AKT in insulin signaling pathway in liver and adipose tissue of mice.Conclusion:The increase of LPS in GDM have an effect on oxidative stress,SIRT1 pathway and insulin signaling,leading to increased inflammatory factors in insulin target organs,subsequently influencing the glucose and lipid metabolism,contributing to insulin resistance,and ultimately participating in the pathogenesis of GDM.This may be associated with the up-regulation of intracellular HDAC1 expression by inflammatory environment and ROS accumulation,which leads to the decrease of NAD~+content,the down-regulation of SIRT1 expression,and the occurrence of insulin resistance. |
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