Cross‐Species Single‐Cell Comparative Analysis Reveals Gluconeogenic Features of Hepatocytes in Ruminants

ABSTRACT In ruminants, hepatic gluconeogenesis supplies approximately 80% of circulating glucose, reflecting a metabolic feature distinct from nonruminants. This pathway supports systemic energy homeostasis and milk production by supplying glucose for lactose synthesis. Although previous studies have described metabolic features and associated gene expression in the ruminant liver, the cellular and regulatory bases of hepatocyte metabolic specialization remain unclear. Here, we constructed a cross‐species single‐cell liver atlas spanning six mammalian species and comprising 101,007 cells. We defined 11 major cell types, among which hepatocytes were further resolved into 10 subpopulations. A ruminant‐enriched hepatocyte subtype, hep1, displayed a pronounced glucose metabolism and gluconeogenic program, characterized by elevated expression of genes such as PFKFB1, PPARGC1A, GYS2, GBE1, AGL, PCCA and PCCB. Integrated transcriptional regulatory network inference, pseudotime trajectory reconstruction, and intercellular communication analyses revealed key transcription factors, including NR3C1, PPARA, NR1H4, and RORA, that may contribute to the ruminant hepatocyte subtype identity. To investigate the regulatory impact of genetic variation on gluconeogenesis‐related genes, we extended these single‐cell insights to the population level in cattle. By performing deconvolution using large‐scale bulk RNA‐seq datasets, we identified expression quantitative trait loci (eQTLs) associated with key metabolic genes (e.g., PPARGC1A and PCCB) in hepatocytes. These findings provide a multidimensional view of the regulatory architecture underlying ruminant liver adaptation and offer insights into the genetic determinants of ruminant metabolic traits.