Nε-(Carboxymethyl)lysine induces mitochondrial stress, inflammatory activation, and fibrogenic remodelling in multicellular human liver organoids.

Abstract

Advanced glycation end-products (AGEs) accumulate in metabolic dysfunction-associated steatotic liver disease (MASLD); however, the contribution of individual AGE species remains poorly defined. We focused on Nε-(carboxymethyl)lysine (CML), a well-characterised AGE generated through glycoxidation and lipid peroxidation, and investigated its association with steatotic liver disease and its effects in multicellular liver models. AGE-RAGE pathway activity was analysed in public liver transcriptomic cohorts, and total AGEs and CML were quantified in human liver and serum. Free CML was then used as a defined experimental stimulus in mice, HepG2 2D/3D, and human induced pluripotent stem cell-derived liver organoid (HLO) models. AGE-RAGE pathway activity was associated with steatohepatitis-related features, whereas hepatic CML was selectively elevated in steatotic liver. Oral CML induced AGE-RAGE- and transforming growth factor beta-associated transcriptional changes in mice but minimal histological fibrosis, whereas HepG2 models showed limited CML-associated lipid accumulation. In contrast, HLOs exhibited concentration-dependent lipid accumulation and fibrogenic remodelling. Single-cell and functional analyses further revealed coordinated mitochondrial stress, inflammatory activation, stellate-cell remodelling, and collagen deposition. Selected responses were attenuated by pathway-targeted interventions, and patient serum cytokine and liver proteomic profiles showed partial concordance with HLO-associated signatures. Together, these findings identify hepatic CML as an AGE-related feature of steatotic liver disease and demonstrate coordinated mitochondrial, inflammatory, and fibrogenic responses to CML in multicellular HLOs. These results support the utility of HLOs for investigating AGE-specific mechanisms in human liver pathology.