Role Of WISP1 In The Pathogenesis Of Insulin Signaling Pathway In Gestational Diabetes Mellitus

Objective: Gestational diabetes mellitus(GDM)is a glucose intolerance disorder with onset or first recognition during pregnancy,which,however,does not meet the criteria of diabetes mellitus for the general population.Hyperglycemia during pregnancy can result in adverse pregnancy outcomes in both fetus and the mother.The pathogenesis of GDM includes insulin resistance and relatively insufficient insulin secretion.There has been increased interest in the role of inflammation as a mediator of metabolic disorders in maternal obesity,which is an important risk factor for the development of GDM.Adipose tissue can modulate the endocrine function by secreting molecules known as adipokines or adipocytokines.Dysfunction of adipose tissue can cause low grade chronic inflammation and insulin resistance.Wnt1-inducible signaling pathway protein-1(WISP1),also known as CCN4,is a member of the extracellular matrix-associated proteins of the CCN family and is a downstream target gene of the canonical Wingless-type(WNT)signaling pathway,and recently determined as a novel adipokine.WISP1 expression is associated with insulin resistance and inflammation in patients with glucose intolerance.The purpose of this study was to investigate the concentration of WISP1 in the serum,and its expression in the abdominal subcutaneous adipose tissue(SAT),and placenta of women with GDM,and to analyze the relationship between WISP1 and obesity,insulin resistance,and chronic inflammation in patients with GDM.In addition,we further explored the role of WISP1 in the pathogenesis of GDM by investigating the effect of WISP1 on lipopolysaccharide(LPS)-induced cell injury in 3T3-L1 adipocytes and its potential mechanism.Methods: Part1: Study 1 population: The subjects were divided into two groups,a GDM group and a normal glucose tolerance(NGT,control)group According to the results of OGTT test at 24-28 weeks of pregnancy.A total of 140 serum samples were collected from Chinese pregnant women with regular follow-up between 25 weeks and 30 weeks of gestation visiting the outpatient department of Shengjing Hospital of China Medical University.Fast glucose,fasting serum insulin and Hb A1 c,thyroid function,triglyceride(TG),high-density lipoprotein cholesterol(HDL-C)and low-density lipoprotein cholesterol(LDL-C),and alanine aminotransferase(ALT)levels were evaluated.Insulin resistance was determined using the HOMA-insulin resistance(HOMA-IR)index.The concentrations of Serum WISP1 and Interleukin(IL)-6 were evaluated with the enzyme-linked immunosorbent assay(ELISA)method.Study 2 population: Tissues of placenta and abdominal SAT samples were obtained from 52 women who had undergone cesarean section,and were divided into a GDM group and a control group.RT-PCR and western blot were used to detect the WISP1 expression.Immunohistochemistry was used to find the localization of WISP1 protein in tissues.Part 2: Lentivirus-mediated overexpression(OE)or knockdown of WISP1 was performed in this study.The glucose consumption test was used to observe the glucose consumption of adipocytes in each group.The effect of WISP1 on the proliferation of adipocytes was observed by MTT assay.To study the effect of WISP1 on LPS-induced cell injury in 3T3-L1 adipocytes,Log-phase 3T3-L1 adipocytes were transiently transfected with the lentivirus.Following transfection,each group was treated with LPS at a concentration of 10 ?g/m L for 24 h.Specifically,the cells were divided into the following groups: Control,LPS,GFP+LPS,WISP1OE+LPS,sh NC+LPS,and sh WISP1+LPS.3T3-L1 adipocytes apoptosis,IL-6 and Tumor necrosis factor(TNF)-? concentration of m RNA and protein were detected by Apoptosis Detection kit,western blotting,ELISA and q RT-PCR respectively in addition to assay the phosphatidyl inositol 3-kinase(PI3K)/ protein kinase B(Akt)pathway activities.Results: 1.The serum WISP1 concentrations were higher in the GDM group than in the control group(P < 0.01)and positively associated with body mass index(BMI),fasting glucose,fasting insulin,HOMA-insulin resistance(HOMA-IR),and IL-6 levels.BMI,fasting glucose,and HOMA-IR independently and positively predicted WISP1 levels.WISP1 m RNA and protein expression were higher in tissues from the placentae and abdominal SAT from the GDM group(P < 0.01).WISP1 protein was localized in both SAT and placental tissue,and the expression level of WISP1 protein in GDM group was higher than that in the control group.2.The expression of WISP1 in 3T3-L1 adipocytes in protein and m RNA in WISP1 OE group were increased significantly compared to GFP group(P<0.05).The MTT results indicated that OD value increased in WISP1 OE group compared with GFP group.LPS promoted cell apoptosis,which significantly upregulated the expression of cleaved-caspase-3 and bax in LPS group(compared to Control group,P<0.05).At the same time,the bcl-2 levels of LPS group(compared with Control group,P<0.05)were significantly down-regulated,which were further confirmed by flow cytometry.Overexpression of WISP1 significantly improved adipocyte apoptosis and reversed the above-mentioned changes in bcl-2,bax,and cleaved-caspase-3 induced by LPS.WISP1 knockdown exhibited the opposite results.WISP1 overexpression promoted the release of inflammatory factors IL-6 and TNF-? and the levels of IL-6 and TNF-? in LPS group and WISP1OE+LPS group were significantly higher than in Control group(LPS group vs Control group)and in GFP+LPS group(WISP1OE+LPS group vs GFP+LPS group)(P<005),while,WISP1 deficiency led to a reduction of IL-6 and TNF-? expression,and the levels of IL-6 and TNF-? in sh WISP1 group were significantly lower than in sh NC+LPS group(P<0.05).the PI3 K and Akt phosphorylation levels in groups LPS,WISP1OE+LPS,and sh NC+LPS were increased greatly as compared to groups Control,GFP+LPS,and sh WISP1+LPS,respectively(P<0.05).But the Fork head box protein O3a(Fox O3a)nuclear translocation was decreased dramatically in groups LPS,WISP1OE+LPS,and sh NC+LPS as compared to Groups Control,GFP+LPS,and sh WISP1+LPS,respectively(P<0.05).In contrast,WISP1 depletion suppressed LPS-induced Akt phosphorylation and promoted Fox O3 a nuclear translocation.After treatment with LY294002(Akt inhibitor),the protective effect of WISP1 on adipocytes was reversed.Conclusion: 1.This study presents novel data showing increased plasma WISP1 levels and increased WISP1 expression in the abdominal SAT and placenta of women with GDM.2.WISP1 can attenuate LPS-induced cell injury of 3T3-L1 adipocytes by regulating the PI3K/Akt signaling pathway.Our current findings may support the hypothesis that WISP1 plays a role in the pathogenesis of GDM.