ORF15-03 - Cytosolic Reductive Stress Drives Hepatic Lipid Remodeling

Background: Reductive stress is a metabolic condition characterized by excessive accumulation of reducing equivalents, manifesting as an increased ratio of NADH to NAD+ (NADH/NAD+). Notably, α-hydroxybutyrate (α-HB), a byproduct of NADH-dependent reactions, was recently identified as a circulating biomarker of hepatic cytosolic reductive stress and is strongly associated with metabolic diseases such as Type 2 diabetes and steatotic liver disease in humans. Although increasingly recognized as a critical regulator of metabolic homeostasis, the mechanisms driving reductive stress-induced metabolic dysfunction remain poorly understood.

Methods: To directly test the metabolic consequences of hepatic cytosolic reductive stress, we expressed E. coli soluble transhydrogenase (EcSTH), a novel metabolic tool that increases the cytosolic NADH/NAD⁺ ratio in mouse liver via adenoviral delivery. Mice were euthanized 4 days after adenovirus injection; hepatic lipidomics was performed to quantify lipid species, and transcriptomics was used to evaluate gene expression changes in the liver. To determine cell-autonomous effects, EcSTH was expressed in AML12 mouse hepatocytes, with or without the co-expression of the NADH-oxidizing enzyme Lactobacillus brevis NADH oxidase (LbNOX), which lowers the cytosolic NADH/NAD⁺ ratio.

Results: Liver-specific EcSTH expression significantly increased hepatic (p < 0.01) and circulating (p < 0.01) triglyceride levels. Hepatic lipidomics revealed increased triglycerides and decreased phosphatidylcholine in the livers of EcSTH mice compared with controls, recapitulating the lipidomic signature observed in patients with steatotic liver disease. Hepatic transcriptomic analysis demonstrated upregulation of de novo lipogenesis (DNL) genes, indicating a redox-driven lipogenic shift. In AML12 hepatocytes, EcSTH expression increased intracellular triglyceride accumulation, confirming a hepatocyte-autonomous effect. Importantly, lowering the cytosolic NADH/NAD⁺ ratio via LbNOX expression reversed triglyceride accumulation, directly implicating reductive stress as the causal signal.

Conclusion: Elevated cytosolic reductive stress reprograms hepatic metabolism by altering lipid species and transcriptional signature of de novo lipogenesis. Given the clinical association of α-HB as a biomarker of impaired metabolic features, including triglyceride levels and glucose intolerance, this work identifies hepatic redox state as a therapeutic target for metabolic diseases.