Integrated multi-omics analysis dissects hepatocyte states and genetic regulation of hepatic metabolism in pigs

Abstract Hepatic metabolism is influenced by both the multi-tissue environment and cellular heterogeneity, yet the cellular basis and genetic regulatory mechanisms underlying liver-specific metabolic functions remain incompletely understood. Pigs share high similarity with humans in liver structure, physiological function, and metabolic characteristics, making them an important large-animal model for studying hepatic metabolic regulation. In this study, we integrated bulk RNA-seq data from 300 liver, spleen, and blood samples with liver scRNA-seq data to characterize pig hepatic metabolic regulation across tissue, cellular, and genetic layers. Multi-tissue transcriptomic analysis identified liver-enriched gene sets mainly associated with metabolic and synthetic functions, highlighting the functional specialization of the pig liver in a multi-tissue context. At single-cell resolution, hepatocytes were further resolved into two major subtypes. Functional annotation, trajectory analysis, and cell–cell communication analysis further showed that these two hepatocyte subtypes displayed distinct functional features: Hepatocytes_Metabolic was enriched for lipid metabolism and core hepatic pathways, whereas Hepatocytes_Immune was more closely associated with immune-related microenvironmental regulation. To investigate the genetic basis of cellular heterogeneity in liver tissue, we incorporated estimated cell-type proportions into cell-type interaction eQTL models. This analysis identified 6,313 ct-ieGenes across liver cell populations, with Hepatocytes_Metabolic showing the largest number of ieGenes (3,404), indicating that the proportion of this metabolic hepatocyte subtype provides an important cellular context for resolving heterogeneity in hepatic genetic regulation. Colocalization analysis further linked ct-ieQTL signals in Hepatocytes_Metabolic to liver-related biochemical and lipid traits, including gamma-glutamyl transferase and cholesterol-related traits. Cross-species analysis showed that pig hepatocytes were transcriptionally closer to human hepatocytes than to mouse hepatocytes, and pig–human conserved hepatocyte genes were mainly enriched in lipid metabolism-related functions. Overall, this study provides a multi-level framework for understanding pig hepatic metabolic regulation and suggests that Hepatocytes_Metabolic represents an important cellular context linking liver metabolic function, cell-composition-associated genetic regulation, and liver-related complex traits.