Diploid Hepatocytes Resist Acetaminophen-Induced Liver Injury Through Suppressed JNK Signaling

Background & Aims: The liver contains both diploid and polyploid hepatocytes, but their functional differences remain poorly understood. Emerging evidence suggests that each ploidy state contributes to regeneration in an injury-specific manner. We hypothesized that diploid hepatocytes promote healing after acetaminophen (APAP)-induced liver injury. Approach & Results: To study ploidy populations in vivo, we utilized mice with a lifelong liver-specific knockout of E2f7/E2f8 (LKO), which are enriched in diploid hepatocytes (>70%) but otherwise normal. Control and LKO mice were treated with APAP (300 or 600 mg/kg), and injury was assessed over 0-96 hours. Although both groups sustained injury, LKO mice showed improved survival, lower serum liver enzyme levels, and reduced necrosis and DNA fragmentation, indicating resistance to APAP-induced injury. To determine if resistance was due to E2f7/E2f8 loss or increased diploidy, we deleted E2f7/E2f8 in adult hepatocytes (HKO), a model that does not alter ploidy. Injury was similar between controls and HKO, ruling out gene deletion as the protective factor. Transcriptomic and protein analyses revealed minimal baseline differences; however, following APAP treatment, LKO livers exhibited reduced JNK activation and less mitochondrial injury. Finally, APAP-treated wild-type hepatocytes exhibited a shift toward lower ploidy, supporting the idea that diploid cells are more resistant to injury. Conclusions: Diploid hepatocytes resist APAP-induced liver injury through reduced JNK activation and mitochondrial damage. These findings highlight hepatocyte ploidy as a key determinant of injury response and suggest a protective role for diploid hepatocytes in promoting liver resilience and regeneration. Overall design: Wild-type (WT) and Alb-Cre–driven liver-specific E2f7/E2f8 knockout mice (LKO; indicated as “KO” in the sample list) were used. Hepatocyte-specific knockouts (HKO; indicated as “CRE” in the sample list) were generated by intravenous injection of AAV8-TBG-Cre viral particles into 2-month-old floxed mice; control mice (indicated as “NULL” in the sample list) received AAV8-TBG-Null. Male, 2.5-month old mice were fasted overnight (~16 hours) and administered 300 mg/kg acetaminophen (APAP) via intraperitoneal injection. Livers were harvested at 0, 12, 24, 48, 72, and 96 hours post-injection and snap-frozen. Total RNA was extracted from frozen liver tissue using TRIzol reagent. For baseline (0 hour) samples, RNA was isolated from individual mice (n = 4 per genotype). For time points from 12 to 96 hours, RNA from three biological replicates per genotype per time point was pooled prior to sequencing (n = 3 mice pooled per group).