Gene-dose effect of the glutathione biosynthesis gene on ascorbate deficiency in mice

Glutathione (GSH) and ascorbate (vitamin C) are key antioxidants with well-established biochemical and clinical interplay in protecting against oxidative stress. Glutamate-cysteine ligase is the rate-limiting enzyme in GSH biosynthesis; its modifier subunit (GCLM) regulates tissue GSH levels. L-gulono-γ-lactone oxidase (GULO) catalyzes a critical step in ascorbate biosynthesis; Gulo-knockout (Gulo^KO) mice, like humans, require dietary vitamin C. Previous work using double-knockout (Gclm^KO/Gulo^KO) mice revealed an essential role of GSH-ascorbate interaction in brain function. Herein, we report an allelic dosage effect of Gclm on redox imbalance and phenotypic outcomes under states of ascorbate deficiency. Gclm^WT/Gulo^KO mice remained overtly healthy with low-ascorbic acid (AA) supplementation at 1.25 mM (mM) in drinking water. In contrast, lacking one copy of the functional Gclm allele (Gclm^HET/Gulo^KO) resulted in increased vulnerability to scurvy development, which was clinically evident with 1.25 mM AA and was mitigated by 2.5 mM AA. Redox profiling revealed insufficient ascorbate retention and a more oxidized glutathione pool in Gclm^HET/Gulo^KO liver and brain tissues at 1.25 mM AA. These results highlight a gene-dose-dependent role of Gclm in maintaining ascorbate homeostasis and redox balance during ascorbate deficiency, with implications for human populations facing limited access to dietary vitamin C and carrying functional GCLM polymorphisms.