Time-of-day-dependent variation of the human liver transcriptome and metabolome is disrupted in MASLD.

Background & Aims: Liver homeostasis is ensured in part by time-of-day-dependent processes, many of them being paced by the molecular circadian clock. Liver functions are compromised in metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH), and clock disruption increases susceptibility to MASLD progression in rodent models. We therefore investigated whether the time-of-day-dependent transcriptome and metabolome are significantly altered in human steatotic and MASH livers. Methods: Liver biopsies, collected within an 8 h-window from a carefully phenotyped cohort of 290 patients and histologically diagnosed to be either normal, steatotic or MASH hepatic tissues, were analyzed by RNA sequencing and unbiased metabolomic approaches. Time-of-day-dependent gene expression patterns and metabolomes were identified and compared between histologically normal, steatotic and MASH livers. Results: Herein, we provide a first-of-its-kind report of a daytime-resolved human liver transcriptome-metabolome and associated alterations in MASLD. Transcriptomic analysis showed a robustness of core molecular clock components in steatotic and MASH livers. It also revealed stage-specific, time-of-day-dependent alterations of hundreds of transcripts involved in cell-to-cell communication, intracellular signaling and metabolism. Similarly, rhythmic amino acid and lipid metabolomes were affected in pathological livers. Both TNFα and PPARγ signaling were predicted as important contributors to altered rhythmicity. Conclusion: MASLD progression to MASH perturbs time-of-day-dependent processes in human livers, while the differential expression of core molecular clock components is maintained. Impact and implications: This work characterizes the rhythmic patterns of the transcriptome and metabolome in the human liver. Using a cohort of well-phenotyped patients (n = 290) for whom the time-of-day at biopsy collection was known, we show that time-of-day variations observed in histologically normal livers are gradually perturbed in liver steatosis and metabolic dysfunction-associated steatohepatitis. Importantly, these observations, albeit obtained across a restricted time window, provide further support for preclinical studies demonstrating alterations of rhythmic patterns in diseased livers. On a practical note, this study indicates the importance of considering time-of-day as a critical biological variable which may significantly affect data interpretation in animal and human studies of liver diseases.

An extended transcription factor regulatory network controls hepatocyte identity.

Cell identity is specified by a core transcriptional regulatory circuitry (CoRC), typically limited to a small set of interconnected cell-specific transcription factors (TFs). By mining global hepatic TF regulons, we reveal a more complex organization of the transcriptional regulatory network controlling hepatocyte identity. We show that tight functional interconnections controlling hepatocyte identity extend to non-cell-specific TFs beyond the CoRC, which we call hepatocyte identity (Hep-ID)CONNECT TFs. Besides controlling identity effector genes, Hep-IDCONNECT TFs also engage in reciprocal transcriptional regulation with TFs of the CoRC. In homeostatic basal conditions, this translates into Hep-IDCONNECT TFs being involved in fine tuning CoRC TF expression including their rhythmic expression patterns. Moreover, a role for Hep-IDCONNECT TFs in the control of hepatocyte identity is revealed in dedifferentiated hepatocytes where Hep-IDCONNECT TFs are able to reset CoRC TF expression. This is observed upon activation of NR1H3 or THRB in hepatocarcinoma or in hepatocytes subjected to inflammation-induced loss of identity. Our study establishes that hepatocyte identity is controlled by an extended array of TFs beyond the CoRC.