Persistent lipotoxicity overwhelms adaptive capacity in human hepatic cell line.

BACKGROUND: Chronic fatty acid (FA) elevation drives metabolic dysfunction-associated liver disease (MASLD). Hepatocytes buffer transient lipid overload via coordinated adaptive mechanisms, but the temporal limits of this capacity remain undefined. OBJECTIVES: We characterized early, intermediate, and prolonged hepatocellular responses to sustained lipid excess. METHODS: HuH7 hepatocytes were exposed to an equimolar palmitic/oleic acid (PA/OA) mixture for 1, 14, or 28 days. Viability, lipid accumulation, stress-response pathways, insulin signaling, metabolic regulators, mitochondrial markers, [14C]-PA uptake, and exo-metabolomic profiles were assessed (n≥3/group). Data (mean ± SEM) were analyzed by two-way ANOVA with Bonferroni post hoc testing; cumulative cell death by linear regression. P<0.05 was significant. RESULTS: FA treatment increased lipid droplet accumulation 1.4- to 1.7-fold vs. time-matched BSA controls at all time points (p<0.001) and increased the rate of cumulative cell death ∼4.3-fold (1.556±0.083 vs. 0.365±0.054%/day, p<0.001). Exo-metabolomic PCA explained 88.5% of variance (Q2=0.758), with peak lactate release at Day 14 and progressively rising acetate through Day 28. [14C]-PA accumulation at Day 28 was ∼30% lower than Day 1 (∼800 vs. ∼500 cpm/mg protein), with a declining rather than plateauing profile between 20-60 s. By Day 28, FA-treated cells showed lower FASN (p<0.001), CD36 (p<0.05), FABP1 (p<0.001), SIRT1 (p<0.005), and CLOCK (p<0.01) protein levels, and higher CPT1A (p<0.001), pAMPK/AMPK (p<0.01), p53 (p<0.001), Bax/Bcl2 (p<0.005), and respiratory complex I-V abundance (p<0.05-p<0.001) vs. controls. CONCLUSIONS: Sustained PA/OA exposure induces a staged hepatocellular response, early lipid-buffering adaptation, intermediate metabolic compensation, and late decompensation, indicating that chronic lipotoxicity reflects progressive loss of regulatory coordination despite persistent metabolic adaptation.