Altered Carnitine Homeostasis Modulates Hyperosmolarity-Induced Mitochondrial Dysfunction and Lipotoxicity in Human Corneal Epithelial Cells

Purpose: Tear hyperosmolarity, a hallmark of dry eye disease (DED), contributes to corneal epithelial injury, inflammation, and mitochondrial dysfunction. This study examined the effects of hyperosmolar stress on mitochondrial carnitine metabolism in human telomerase-immortalized corneal epithelial (hTCEpi) cells and evaluated the osmoprotective potential of L-carnitine supplementation. Methods: The hTCEpi cells were exposed to 90 mM NaCl to induce 490 mOsM hyperosmolar conditions, with or without 200 mM L-carnitine supplementation. Free fatty acid (FFA) uptake, triglyceride (TG) accumulation, lipid peroxidation, and mitochondrial reactive oxygen species (mtROS) were quantified using fluorometric and colorimetric assays. The expression and activity of carnitine palmitoyltransferases (CPT1 and CPT2), carnitine acetyltransferase (CrAT), and peroxisome proliferator-activated receptor alpha (PPARα) were assessed, and intracellular ATP levels were measured as indicators of mitochondrial function. Results: Hyperosmolar stress markedly increased lipid uptake, TG accumulation, malondialdehyde (MDA) levels and mtROS generation. CPT1, CPT2, and CrAT expression and activity and ATP levels were decreased, whereas inducible nitric oxide synthase (iNOS) expression was upregulated. L-carnitine supplementation significantly reduced lipid accumulation and peroxidation, decreased mtROS production, and partially restored ATP generation. It also enhanced total CPT and CrAT enzyme activities, indicating recovery of carnitine shuttle function. Conclusions: These findings demonstrate that hyperosmolarity disrupts carnitine homeostasis, and leads to lipotoxicity, oxidative stress, and reduced ATP production. In contrast, L-carnitine supplementation restores carnitine-dependent mitochondrial pathways and may represent a potential therapeutic strategy to protect the ocular surface from hyperosmolar injury in DED.