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.