Mechanism Of Abnormally Elevated BCAA In Pregnancies With Advanced Maternal Age Leads To GDM

Background:Gestational diabetes mellitus(GDM)is a unique glucose intolerance in pregnancy,which can be harmful to maternal and neonatal outcomes.It can contribute to adverse neonatal outcomes and maternal long-term type 2 diabetes and cardiovascular diseases.Obesity,overnutrition,advanced maternal age,metabolism and genetic factors can predispose to the onset of GDM.In recent years,the incidence of GDM increases with the elevation of pregnancies with advanced maternal age(≥35 years old)resulting from the change of fertility policy.The underlying pathogenesis involves insulin resistance(IR),the relative insufficiency of insulin secreted by pancreatic isletβcells and oxidative stress.Metabolism of the three major substances in humans affects each other.Abnormal blood glucose can lead to changes in amino acid metabolism during pregnancy,and vice versa.Rapid developments in metabolomics including GC-MS and LC-MS have made it available to detect small molecule metabolites and discover various biomarkers for GDM including branched-chain amino acids(BCAA).BCAA refers to a class of amino acid with similar branch chains onα-carbon,including leucine,isoleucine and valine,which are essential amino acids for human.High concentrations of BCAA can lead to oxidative stress.Currently,elevated circulating BCAAs have been found in type 2 DM and cardiovascular diseases.However,the association between BCAA and GDM is still unclear,so this study aims to explore whether BCAA can cause GDM and the potential mechanism.Methods:1.Analysis of clinical sample data:20 elderly GDM pregnancies(more than 35 years old)and 20 elderly healthy pregnancies were recruited.The plasma metabolomic profiles were detected by GC-MS in the early(11-14 weeks)and the third(32-34 weeks)trimester.2.Establishing animal model for GDM and conducting serum metabolomic analysis:12 months old C57BL/6J mouse were fed with high fat diet and low fat diet respectively(n=6 per group)for one week before mating.Time of presence of vaginal plugs was recorded as GD0.5.On GD0.5,GD11.5and GD 16.5 days,oral glucose tolerance test was taken to investigate the differences in glucose tolerance between the two groups.On GD18.5,the fasting blood glucose was detected by glucose oxidase and the serum fasting insulin levels were detected by enzyme-linked immunosorbent assay after sacrificing the mouse.Homeostasis model assessment-Insulin Resistance was calculated.The serum,liver,muscle and fat tissues were collected.Serum metabolomic profiles of mouse were detected by GC-MS.3.The serum metabolomic profiles of human skeletal muscle cells were detected after treatment with BCAA(10mmol/L)for 24 hours by GC-MS.4.DCFH-DA probe was performed to detect ROS levels in human skeletal muscle cells after treatment with different concentrations of BCAA(2mmol/L,4mmol/L,6mmol/L,8mmol/L,10mmol/L)for 24 hours.5.Western blot(WB)assay was used to detect the expression of NOX4,p-JNK,JNK,PI3K,p-Akt,Akt,serine phosphorylated IRS-1,SIRT1,GLUT4 in human skeletal muscle cells after BCAA treatment.6.The Seahorse XF Energy Metabolism Detection System measures mitochondrial respiration of L02 cells and human skeletal muscle cells after treatment with different concentrations of BCAA(4mmol/L,6mmol/L,8mmol/L,10mmol/L,12mmol/L)for 24 hours.Results:1.There were 14 and 39 significant different metabolites in plasma detected by GC-MS in the first and third trimester respectively between GDM and normal control group.The plasma BCAA level in the GDM group was increased.2.On GD11.5 and GD16.5,there were significant differences in glucose tolerance between HFD mice and LFD mice.The mice model for GDM was successfully established.On GD18.5,there were significant differences in fasting glucose,fasting insulin levels and HOMA-IR between the two groups;there were 22 significantly different metabolites in the serum of the two mice groups,especially BCAA levels.3.65 significantly different metabolites were found by GC-MS after treatment with 10mmol/L BCAA compared with normal group.KEGG pathway analysis revealed significant differences in oxidative stress related pathways between the BCAA group and the control group.4.Intracellular ROS was elevated after BCAA treatment and reached the highest level in human skeletal muscle cells at the concentration of 10mmol/l.5.The expressions of NOX4,serine phosphorylation IRS-1/IRS-1,p-JNK/JNK in BCAA-treated group were increased compared with normal control group,while the expressions of PI3K,p-Akt/Akt,SIRT1 and GLUT4 were decreased.6.The Seahorse XF Energy Metabolism Detection System showed decreased basal respiration,maximum respiration,ATP production and H~+leakage in L02 cells and human skeletal muscle cells.Conclusions:Circulating BCAA levels in elderly GDM pregnancies and animal models were increased.Cell experiments and molecular biology showed that the expressions of oxidative stress associated proteins were increased,while the expressions of insulin signaling pathway were inhibited in insulin-sensitive cells after BCAA treatment.Therefore,our study suggests that abnormally high BCAA level may promote the occurrence and development of GDM and provide a new strategy for the prevention and treatment of GDM in the future.