Time-Dependent Phospholipid Remodeling in Cultured Primary Mouse Hepatocytes: Associations with PEMT Status and Methionine Availability.

BACKGROUND: Cultured primary mouse hepatocytes undergo drastic phenotypic and metabolic reprogramming, while the temporal rules and regulatory machinery of membrane phospholipid remodeling remain elusive. METHODS: Relying on a 0-72 h time-series in vitro culture system, this study integrated multi-omics technologies to dissect the temporal dynamics of phospholipid remodeling in hepatocytes. Through phosphatidylethanolamine N-methyltransferase (PEMT) knockout, exogenous PEMT expression, and methionine deprivation, we examined the association of PEMT status and methionine availability with phospholipid remodeling. RESULTS: In vitro cultivation reduces intracellular total phospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) through three coordinated events: suppressed transcription of phospholipid synthetic genes hinders de novo synthesis, elevated lipid hydrolysis consumes cellular phospholipids, and extracellular phospholipids accumulate in the culture medium from 12 to 48 h. These jointly trigger ordered remodeling of PC/PE balance, acyl chain length and fatty acid unsaturation. PEMT knockout was associated with PE retention without worsening hepatocyte dedifferentiation, PEMT exogenous expression raises PC content and PC/PE ratio yet cannot rescue culture-dominated lipid structural shifts. Methionine depletion depleted cellular methionine, S-adenosylmethionine (SAM) and S-adenosyl-L-homocysteine (SAH), producing selected lipid changes that partially overlapped with lipid phenotypes of PEMT knockout. CONCLUSION: In short, culture duration was the dominant factor associated with the fundamental phospholipid remodeling trajectory, and PEMT status and methionine availability, were associated with selective differences in lipid composition.