Persistent lipotoxicity overwhelms adaptive capacity in human hepatic cell line

Background: Chronic fatty acid (FA) elevation drives metabolic dysfunction-associated steatotic liver disease. 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 d. Viability, lipid accumulation, stress-response pathways, insulin signaling, metabolic regulators, mitochondrial markers, [14C]-PA uptake, and exometabolomic profiles were assessed (n ≥ 3/group). Data (mean ± standard error of the mean) were analyzed by 2-way analysis of variance with Bonferroni post hoc testing; cumulative cell death was analyzed by linear regression. P < 0.05 was considered significant. Results: FA treatment increased lipid droplet accumulation 1.4- to 1.7-fold compared with time-matched bovine serum albumin controls at all time points (P < 0.001) and increased the rate of cumulative cell death ∼4.3-fold (1.556% ± 0.083% compared with 0.365 ± 0.054%/d, P < 0.001). Exometabolomic principal component analysis 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 compared with ∼500 cpm/mg protein), with a declining rather than plateauing profile between 20 and 60 s. By day 28, FA-treated cells showed lower FA synthase (P < 0.001), CD36 (P < 0.05), FABP1 (P < 0.001), SIRT1 (P < 0.005), and CLOCK (P < 0.01) protein levels, and higher carnitine palmitoyl transferase 1A (P < 0.001), phosphorylated AMP-activated protein kinase (AMPK)/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) compared with 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.