Multi-omics approaches to characterize acute and cyclic fasting responses

Abstract

Fasting activates a coordinated systemic response involving key metabolic tissues, like adipose tissue (AT) and liver. At the onset of fasting, AT rapidly mobilizes stored lipids, while the liver initiates glycogenolysis followed by gluconeogenesis and ketogenesis to maintain long-term energy homeostasis. Key players in these adaptations are transcription factors (TFs) that bind to promoter and enhancer regions to coordinate cellular transcriptional programs. Although individual fasting-responsive regulators have been described, their time-resolved coordinated activity remains not fully understood. Transcriptionally active TF-bound enhancers generate short-lived enhancer RNAs (eRNAs). Using quantitative precision run-on sequencing (qPRO-seq) we generated the first nascent transcription-based atlas of fasting-regulated genes and enhancers in mouse liver and AT. In AT, we observed a rapid activation of lipolytic genes (Pnpla2, Hsl) and downregulation in lipid storage genes (Gk) within 3-6 hours after food withdrawal. Using qPRO-seq thousands of fasting-selective eRNAs and super enhancers were identified, showing dynamic enhancer activation. TF foot printing in those enhancers highlighted early activation of TFs such as SP1, NFE2L2 and RARγ. Resolving the cell-specific transcriptional landscape by integrating qPRO-seq with snATAC-seq uncovered fasting induced remodeling in adipocytes at genes implicated in lipid metabolism (Nrf1) and autophagy (Atg7) after 6 hours. A fasting time-line analysis in liver with qPRO-seq revealed temporally structured transcriptional activation of key metabolic genes, including Pck1 and G6pc. Enhancer profiling unravelled progressive TF motif complexity over time, with early enrichment of chromatin-structural (CTCF) and circadian (REV-ERB, NPAS2, BMAL1) TF motifs, followed by hepatic metabolic and immune regulators (FXR, NR5A2, IRF, BATF, NF-kB). After 24 hours, enhancers were enriched for canonical fasting TFs, including PPAR, FoxO and C/EBP family members. Finally, we compared the systemic and hepatic responses to short- and long-term dietary restriction in mice subjected to either intermittent fasting (IF) or fasting-mimicking diet (FMD). Both interventions activated PPARα-driven fatty acid oxidation and ketogenesis in the liver. Short-term FMD (one cycle) induced a stronger metabolic response than IF, which was reflected by higher levels of plasma FGF21 and ketone bodies. Notably, while repeated IF (4 cycles) preserved fatty acid oxidation repeated FMD (3 cycles) supressed it, fatty acid oxidation, which likely reflects fat store depletion. Together, our integrative multi-omics analysis revealed time-resolved responses on transcriptional and chromatin landscapes in AT and liver during acute and cyclic fasting. We identified fasting-induced (super-) enhancers and TF networks that drive the adaptation to fasting. Thus, our study represents a valuable resource for future studies to understand the fasting response on the molecular level, potentially yielding refined preventive and therapeutic fasting protocols for humans. 

Datum der Bewilligung	2025
Originalsprache	Englisch
Gradverleihende Hochschule	Medizinische Universität Graz
Betreuer/-in	Jelena Krstic (Betreuer*in), Simon Sedej (Betreuer*in) & Andreas Prokesch (Betreuer*in)