Sustained Hyperglycemia Drives Hepatic Metabolic Rewiring and Is Selectively Modulated by a Probiotic Strain in a Chicken Embryo Model of GDM

Background Gestational diabetes mellitus (GDM) exposes developing embryos to sustained hyperglycemia, yet dissecting embryo‑intrinsic mechanisms remains difficult in mammalian models due to maternal and placental influences. To directly evaluate hyperglycemia‑driven metabolic programming and probiotic modulation, we established a placenta‑independent in ovo chicken embryo model and tested the probiotic Leuconostoc pseudomesenteroides (MLS3) in live and heat‑inactivated (HI) forms. Methods and results Sustained hyperglycemia was induced by repeated air‑sac administration of D‑glucose from embryonic day (ED) 4–18, elevating circulating glucose, reducing embryonic mass, and increasing relative liver weight. Co‑administration of MLS3 attenuated systemic glycemia in a form‑dependent manner, with live MLS3 producing the greatest reduction, and preserved overall growth. Histological evaluation showed hepatic lipidosis and treatment‑specific glycogen accumulation without necroinflammation. Despite steatogenic features, hepatic SOD1 expression and malondialdehyde concentrations remained unchanged, consistent with an ED18 metabolic reliance on β‑oxidation and strong antioxidant buffering. Transcriptomic profiling revealed broad perturbations in oxidative phosphorylation, glycolysis/gluconeogenesis, and pyruvate metabolism, including robust induction of LDHA and PCK1, indicating a dual high‑flux metabolic state. Probiotic effects were highly context dependent: live MLS3 with glucose normalized TLR2/TLR4 signaling, suppressed lipogenesis, restored β‑oxidation, recalibrated ATP‑synthase stoichiometry, and markedly enhanced gluconeogenesis. HI MLS3 conferred partial glycemic benefit but maintained glycolytic and lipogenic signatures with a stress‑skewed transcriptome. Conclusion This embryo‑intrinsic model identifies a hyperglycemia‑induced dual metabolic program and demonstrates that live MLS3 elicits coordinated immunometabolic and mitochondrial rescue. These findings support strain‑informed probiotic strategies and provide a tractable platform for mechanistic studies preceding mammalian GDM models.