Study On Isoforms Of RBP4 And The Pathogenesis Of Gestational Diabetes Mellitus

Gestational diabetes mellitus(GDM),which is defined as impaired glucose tolerance that first appears during pregnancy.The prevalence of GDM is reportedly as high as 17.5%in China.GDM not only increases maternal perinatal complications and complications,the risk of developing type ? diabetes after delivery is more than seven times higher than that of pregnant women with normal glucose tolerance during pregnancy.GDM is also a risk factor of future metabolic syndrome,non-alcoholic fat liver disease,obesity and cardiovascular disease.Moreover,there is a substantially increased risk of obesity,insulin resistance,type ? diabetes(T2DM)and cardiovascular disease in offspring of GDM due to aberrant Intrauterine environment,developing a terrible cycle of diabetes.To study the mechanism of GDM,explore possible ways of blocking,and find therapeutic targets will not only help reduce the incidence of T2DM,but also affect the survival of offspring,directly affecting the quality of the entire population.Pathological insulin resistance during pregnancy is one of the main pathogenesis of GDM.A large number of evidence-based medical evidence shows that pre-pregnancy obesity and excessive weight gain during pregnancy are both independent risk factors for GDM,Overweight during pregnancy leads to an increase in adipose tissue.Adipose tissue is not only an energy storage organ,more importantly,it has endocrine function.Adipokine secreted by adipocytes can participate in the regulation of various metabolic processes through direct or indirect action.The view that Adipokine is an indispensable link between obesity and systemic insulin resistance has reached an agreement.Retinol Binding Protein-4(RBP4),which is responsible for binding and transporting vitamin A in plasma,is a novel adipose-derived cytokine,and plays an important role in obesity-induced insulin resistance in diabetes.Many clinical studies have found that serum RBP4 levels are associated with insulin resistance and positively correlated with BMI and triglycerides in people with obesity and impaired glucose tolerance and patients with T2DM.Exercise therapy and pioglitazone can reduce serum RBP4 levels and improve insulin resistance.Although several studies about change of RBP4 in GDM have been published,there are still some controversies about the correlation between RBP4 levels and GDM.So far,most of the studies between RBP4 and GDM focus on serum total RBP4 level,while ignoring two different forms of RBP4,including retinol-bound RBP4(holo-RBP4)and retinol-free RBP4(apo-RBP4).The physiological role of RBP4 is to bind and transport vitamin A to target tissues.Holo-RBP4 transformed to free apo-RBP4 state after releasing retinol.It has been reported that the ratio of circulating RBP4 and retinol in patients with T2DM and impaired glucose tolerance is higher than individuals with normal glucose tolerance,and the RBP4/retinol ratio can better evaluate the state of glucose metabolism than total RBP4,which imply that apo-RBP4 may participate in abnormal glucose metabolism.Abnormal increase of apo-RBP4 has also been reported in obese patients.Therefore,we suggest that difference conclusions between RBP4 and insulin resistance may be due to the existence of two isoforms of RBP4.According to the reported mechanism that RBP4 participates in regulating insulin signaling pathway,combined with the abnormal expression of two isoforms of RBP4 in metabolic diseases,we speculate that abnormal elevation of apo-RBP4 inhibits insulin signaling pathway and then induces insulin resistance which contributes to GDM.Our study intends to examine the serum RBP4 and retinol concentrations in GDM and normal pregnant women and detect clinical indicators related to insulin resistance,also to analyze the correlation between RBP4's existing forms and metabolic indicators.At the same time,to determine whether change of RBP4/retinol ratio affects insulin signaling pathway through animal and cell models.This study is helpful to elucidate the molecular mechanism of RBP4 in the developing of GDM,find out the factors that predict the early onset of GDM,carry out early intervention to reduce the incidence of maternal-fetal complications caused by GDM.Part ? Isoforms of RBP4 in Gestational Diabetic Patients and its Clinical SignificanceAimsTo investigate the two isoforms of serum RBP4 and their correlation with clinical indicators in pregnant women,and expression of RBP4 in abdominal adipose tissue in patients of gestational diabetes mellitus(GDM).MethodsSeventy-four singleton pregnant women with GDM and 74 singleton pregnant women with normal glucose tolerance who had elective cesarean section were included in the present study.All subjects had regular prenatal tests and were full-term at delivery.Fasting blood was collected from pregnant women before cesarean section,and a small amount of abdominal adipose tissue was collected during the operation.The clinical data of two groups during pregnancy were also recorded.Fasting blood glucose(FBG)was measured with the hexokinase method.Serum total cholesterol(TC),triglyceride(TG),low density lipoprotein(LDL)and high density lipoprotein(HDL)concentrations were measured by enzyme assay.Fasting insulin(FINS),RBP4 and retinol were measured by enzyme-linked immunosorbent assay(ELISA).The relative expression of RBP4 in abdominal adipose tissue was measured by Western Blot.Correlation analyses between two parameters were evaluated using the Spearman correlation analysis.To evaluate the contribution of different variables to total RBP4,RBP4/retinol ratio and apo-RBP4,multivariate linear regression analyses were performed,using stepwise selection.Results1.The maternal age of the GDM group was significantly higher than that of control group(34.6 ± 4.5 vs 32.5 ± 3.7,P<0.01).Compared with normal pregnant women,patients with GDM had higher pre-pregnancy weight(57.0[interquartile range 51.0-62.6]vs 52.6[50.0-57.9]kg,P<0.01),pre-pregnancy BMI(22.1[20.0-24.3]vs 20.7[19.0-22.1]kg/m2,P<0.01),BMI at delivery(27.3[25.4-29.4]vs 26.4[24.8,28.1]kg/m2,P<0.05).GDM patients had significantly higher birthweight(3450[3300-3812]vs 3385[3050-3550]g,P ? 0.05),FBG(4.33 ± 0.66 vs 3.87 ± 0.52 mmol/L,P<0.05)and triglyceride(3.73[2.92-4.55]vs 3,19[2.58-3.87]mmol/L,P<0.05)compared to those with normal glucose tolerance.The level of LDL cholesterol was significantly lower in women with GDM than in those with normal glucose tolerance(2.65[2.30-3.47]vs 3.07[2.61-3.67]mmol/L,P<0.05).2.There were no significant differences in serum RBP4,retinol or RBP4/retinol ratio between the two groups.3.The serum RBP4 was positively correlated with FINS(rs = 0.240,P<0.05),HOMA-IR(rs = 0.222,P<0.05)and triglyceride(rs = 0.200,P<0.05)in the pooled sample.The RBP4/retinol ratio was positively correlated with FINS(rs = 0.255,P<0.05),HOMA-IR(rs = 0.238,P<0.05)and total cholesterol(rs = 0.165,P<0.05).The apo-RBP4 was positively correlated with FINS(rs = 0.246,P<0.05),HOMA-IR(rs =0.230,P<0.05)and total cholesterol(rs = 0.163,-P<0.05).In multiple linear regression analysis,Total RBP4(standardized Beta = 0.172,95%CI 0.011-0.333,P =0.037),RBP4/retinol ratio(standardized Beta = 0.191,95%CI 0.029-0.351,P = 0.021)and apo-RBP4(standardized Beta = 0.191,95%CI 0.011-0.333,P = 0.020)remained independently and positively associated with FINS after adjusting for BMI and lipids.4.The relative expression of RBP4 in abdominal adipose tissue of GDM patients was higher than that of controls(0.57 ± 0.03 vs 0.39 ± 0.02).Conclusion1.There are no significant differences in serum RBP4,retinol or RBP4/retinol ratio between the two groups,but the relative expression of RBP4 in abdominal adipose tissue of GDM patients is higher than that of controls.2.Total RBP4,RBP4/retinol ratio and apo-RBP4 remain independently and positively associated with FINS after adjusting for BMI and lipids.Part II Effects of apo-RBP4 and holo-RBP4 on insulin signaling pathway in pregnant rats and primary adipocyteAimsTo investigate the effect of change of RBP4/retinol ratio on systemic insulin resistance through pregnant rat model;To determine the molecular mechanism of apo-RBP4 and holo-RBP4 on regulation of insulin signaling pathway by primary adipocytes.MethodsNormal Sprague-Dawley pregnant rats were divided into two groups to receive either recombinant apo-RBP4 protein or PBS injection.The serum RBP4 and retinol levels were evaluated after injection.FBS,FINS,HOMA-IR and insulin tolerance test(ITT)were used to assess insulin sensitivity.White adipose tissue,skeletal muscle,and liver tissue were collected to assess the phosphorylation of insulin receptor(IR)and insulin receptor substrate-1(IRS-1)by western blotting.The recombinant protein of apo-RBP4 and holo-RBP4 were prepared and purified.Primary human adipocytes were cultured in vitro with different proportions of apo-RBP4 and holo-RBP4 for 24 h.The interaction between RBP4 and STRA6 was assessed by co-immunoprecipitation,and the expression of JAK-STAT pathway,SOCO3,PPAR? and insulin signaling were detected by western blotting.The cellular localization of GLUT4 was detected by immunofluorescence.Results1.Injection of apo-RBP4 into normal pregnant rats elevated the serum RBP4 level and RBP4/retinol ratio but did not significantly affect the serum retinol concentration.2.The apo-RBP4-treated group showed an increase in blood glucose levels at 15,30,and 60 min compared with control rats.Compared with controls,the levels of FBG,FINS,and HOMA-IR in apo-RBP4-injected rats were markedly higher.3.In vitro,a prolonged interaction between RBP4 and STRA6 was observed when apo-RBP4 was present.In response to increased apo-RBP4 levels,cells showed elevated activation of the JAK2/STAT5 cascade and SOCS3 expression,decreased phosphorylation of IR and IRS1,and attenuated GLUT4 translocation and glucose uptake upon insulin stimulation.ConclusionApo-RBP4 is a ligand that activates the STRA6 signaling cascade,inducing insulin resistance in GDM.