Selective ablation of p16^Ink4a-lineage hepatocytes attenuates hepatic steatosis in diet-induced MASH

The cyclin-dependent kinase inhibitor p16^Ink4a is a widely used marker of cellular senescence; however, the contribution of p16^Ink4a-expressing hepatocytes to hepatic steatosis in metabolic dysfunction-associated steatohepatitis (MASH) remains unclear. Here, we combined p16-CreER-tdTomato lineage tracing, single-cell transcriptomic analysis, and intersectional hepatocyte-specific ablation using a diet-induced MASH model. The NASH2 diet increased the proportion of p16^Ink4a-lineage-positive [p16-Lin(+)] cells among hepatocytes from approximately 0.4% to 2.0% without significantly altering their proportions among cholangiocytes, endothelial cells, macrophages, or hepatic stellate cells. Single-cell RNA sequencing further showed that hepatocytes constituted 76.5% of the p16-Lin(+) population, compared with 34.4% of the p16-Lin(-) population. Despite their low abundance, selective ablation of p16-Lin(+) hepatocytes reduced hepatic lipid accumulation by approximately 50% (P < 0.0001) without altering the F4/80-positive macrophage area. Hepatocyte re-clustering identified a p16-Lin(+)-enriched hepatocyte subcluster with a transcriptional profile consistent with a zone 2 identity, whereas lipid accumulation occurred predominantly in the pericentral zone 3 region. Within this subcluster, p16-Lin(+) hepatocytes showed increased expression of multiple genes encoding secreted proteins, including complement- and coagulation-associated factors, and the chemokine Cxcl12. Together, these findings demonstrate that a small population of p16-Lin(+) hepatocytes contributes disproportionately to hepatic steatosis, and suggest that secreted factors may mediate their effects on surrounding hepatocytes. Thus, the selective targeting of this population may provide a strategy for limiting hepatic lipid accumulation in MASH.