Legumain level in patients with gestational diabetes to promote ferroptosis through liver gluconeogenesis by HSP90 and GPX4.

Background: The incidence of gestational diabetes mellitus (GDM) is the first diabetes in pregnancy and has gradually increased worldwide, increasing the burden of social healthcare systems. In GDM, oxidative stress induced by reactive oxygen species (ROS) can disrupt the integrity of the cell membrane through lipid peroxidation reactions and trigger ferroptosis, further exacerbating pancreatic islet β-cell dysfunction and insulin resistance. Lipid peroxidation products related to ferroptosis (such as malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE)), along with the decrease in glutathione peroxidase 4 (GPX4) activity, may be involved in the placental oxidative damage and adverse fetal outcomes in GDM, suggesting that targeting the antioxidant pathway or regulating ferroptosis may serve as an intervention strategy. Legumain (LGMN) can serve as novel targets in diabetes mellitus genetic therapy. Objective: This study investigated the mechanism and effects of LGMN in GDM. Method: All blood samples of normal or patients with GDM were collected by Beijing Ditan Hospital Affiliated Capital Medical University. C57BL/6J female mice were intraperitoneally injected with streptozotocin. Sh-LGMN virus (20 μg of each) or control vector virus (20 μg of each) was injected into GDM mice. GDM mice randomly assigned to three groups (Number = 10). Sh-LGMN virus (20 μg of each) + HSP90 inhibitor or Sh-LGMN virus (20 μg of each) or control vector virus (20 μg of each) was injected into GDM mice. LGMN or si-LGMN Plasmids were transfected into HepG2 cells using Lipofectamine 2000. HepG2 cells were incubated by different insulin concentrations (100 nmol/L) treatment for 24 h. Microarray analysis, quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, proliferation assay, ethynyl deoxyuridine staining, bioluminescence imaging, and Western blot were used in this study. Results: Serum LGMN mRNA expression was significantly elevated in patients with GDM. LGMN mRNA and protein expression were also elicited in the liver tissue of GDM mice. LGMN mRNA expression exhibited a positive correlation with Body Mass Index (BMI), fasting plasma glucose, 1-h plasma glucose, or 2-h plasma glucose in patients. Sh-LGMN virus reduced blood glucose levels and body weight, inhibited fasting insulin (FINS) levels and Homeostatic Model Assessment of Beta-cell function (HOMA-β), enhanced Fasting Blood Glucose (FBG)/FINS/Total Cholesterol/Triglycerides levels, improved hepatic fibrosis (HE staining), and also upgraded HbAIc and HOMA-IR in mice of GDM. LGMN exacerbated ROS-induced oxidative stress in the in vitro model of GDM. LGMN promoted ferroptosis in vitro model of GDM. LGMN expanded ROS-induced mitochondrial damage in vitro model of GDM. LGMN inhibited the HSP90/GPX4 Signaling Pathway in the model of GDM. The inhibition of HSP90 reduced LGMN on GDM in the mice model or the in vitro model of GDM. LGMN interlinked with complex protein body of HSP90 and GPX4, which LGMN inhibited the HSP90/GPX4 Signaling Pathway. Conclusions: The LGMN level in patients with GDM was upregulated, and LGMN facilitates ferroptosis by HSP90 and GPX4 in the mice model of GDM and may lead to therapeutic potential of ferroptosis or liver gluconeogenesis in the model of GDM.