Science Advances · 21 authors, 14 centres
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This study used mice whose livers were repopulated with human hepatocytes to show that peripheral organ clocks can influence the central circadian pacemaker. Liver humanization advanced the phase of the liver clock, muscle clock, and whole-body rhythms, and shortened the free-running period. These findings reveal a feedback loop from peripheral tissues to the brain's master clock, with implications for understanding circadian disruption in metabolic and liver diseases.
**Background:** The circadian system is hierarchically organized, with a central clock in the suprachiasmatic nuclei (SCN) synchronizing peripheral clocks. While the SCN is known to coordinate peripheral tissues, whether peripheral clocks can feedback to influence the central pacemaker is poorly understood. Previous studies disrupting the hepatocyte clock in mice found no impact on the SCN, and restoring Bmal1 only in hepatocytes of global Bmal1 knockout animals did not restore behavioral rhythms. This study used a chimeric mouse model with humanized livers to test whether hepatocytes with different circadian properties can affect the central clock.
**Methods:** The authors used FRGN mice (Fah-/- Rag2-/- Il2rg-/- on NOD background) that allow >70% liver repopulation with transplanted hepatocytes. Liver-humanized mice (LHM) received primary human hepatocytes; control liver-“murinized” mice (LMM) received murine hepatocytes. Only LHM with circulating human albumin >3500 μg/ml (corresponding to >70% repopulation) were used. Mice were housed under 12h light/12h dark cycles with ad libitum food. For transcriptomic analysis, tissues (liver, quadriceps muscle, SCN, arcuate nucleus) were collected every 4 hours over 24h (ZT2,6,10,14,18,22). RNA-seq reads were mapped to a chimeric mouse-human genome, and rhythmicity was assessed using the dryR method. Metabolic phenotyping was performed using indirect calorimetry (Phenomaster) to measure locomotor activity, food intake, respiratory exchange ratio (RER), and fat oxidation. Circadian period was determined by recording wheel-running activity in constant darkness for 2 weeks. A daytime feeding challenge (food only from ZT0 to ZT12 for 7 days) tested entrainment flexibility.
**Key Results:** In the liver, the largest group of rhythmic genes (model 4) lost rhythmicity in LHM, including genes involved in protein synthesis and mTOR pathway. Phosphorylation of ribosomal protein S6 (a TORC1 target) confirmed loss of rhythmic mTOR activation in LHM. Human hepatocytes showed blunted response to growth hormone (GH): mouse GH was high while IGF-1 was low in LHM, and sex-biased gene expression was perturbed. Importantly, 110 genes (model 14) showed a phase advance in both human and mouse hepatocytes in LHM compared to LMM, including most circadian clock genes (e.g., Arntl, Per2, Nr1d1). In muscle, 13% of rhythmic genes (model 5) showed altered phase, with all clock genes phase-advanced in LHM. In the SCN and arcuate nucleus, 30% and 26% of rhythmic genes (model 3) lost rhythmicity in LHM, respectively, enriched for ion transport genes. A small subset (2% SCN, 10% ARC) showed altered phase, including Nr1d1 and Dbp. Behaviorally, LHM exhibited an approximately 2-hour phase advance in locomotor activity, food intake, RER, and fat oxidation under light/dark cycles. The circadian period in constant darkness was significantly shorter in LHM (mean period not explicitly stated in the provided text, but Fig. 4K shows a significant difference). During daytime feeding, LHM shifted ~90% of drinking activity to the light phase within 3 days, whereas LMM maintained most drinking at night, indicating faster entrainment.
**Clinical Implications:** This study provides direct evidence that peripheral hepatocyte clocks can feedback to the central SCN pacemaker, altering circadian period, phase, and entrainment properties. The findings challenge the strict hierarchical model of circadian organization and suggest that peripheral signals (e.g., from the liver) can act as potent Zeitgebers. This has implications for understanding circadian disruption in metabolic diseases (e.g., nonalcoholic fatty liver disease, cirrhosis) where liver function is impaired and patients often exhibit sleep disturbances and altered hormonal rhythms. The results also suggest that therapies targeting peripheral clocks might influence central circadian regulation. The chimeric model offers a unique tool to study species-specific differences in circadian physiology and the liver-brain axis.