Single-cell metabolic profiling reveals subgroups of primary human hepatocytes showing heterogeneous responses to drug challenge.

Xenobiotics are primarily metabolized by hepatocytes in the liver, and primary human hepatocytes (PHHs) are the gold standard model for the assessment of drug efficacy, safety and toxicity in the early phases of drug development. Recent advances in single-cell genomics have shown liver zonation and ploidy as main drivers of cellular heterogeneity. However, little is known about the impact of hepatocyte specialization on liver function upon metabolic challenge, including hepatic metabolism, detoxification, and protein synthesis. Here, we investigate the metabolic capacity of individual human hepatocytes in vitro, and assess how chronic accumulation of lipids enhances cellular heterogeneity and impairs the metabolisms of drugs. A phenotyping five-probe cocktail was used to identify four functional subgroups of hepatocytes that respond differently to drug challenge and fatty acid accumulation. These four subgroups display differential gene expression profiles upon cocktail treatment and xenobiotic metabolism-related specialization. Notably, intracellular fat accumulation leads to increased transcriptional variability and diminished the drug-related metabolic capacity of hepatocytes. Our results demonstrate that, upon a metabolic challenge such as exposure to drugs or intracellular fat accumulation, hepatocyte subgroups lead to different and heterogeneous transcriptional responses.