Persistent lipotoxicity overwhelms adaptive capacity in human hepatic cell line.
TL;DR
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.
Abstract
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.