Experimental and mathematical models reveal that AMPK modulates circadian clock gene expression and period through NAD+-dependent regulation of Bmal1
This study reveals that AMPK activation by AICAR can influence circadian clock gene expression, but the implications for human health and clinical practice are not yet established.
Where it sits
this study against the rest of the aicar (acadesine) corpusSummary and findings
The study investigates the role of AMP-dependent kinase (AMPK) in regulating circadian clock gene expression, specifically Bmal1, using human U2OS cells. AICAR was used to pharmacologically activate AMPK, resulting in increased Bmal1 promoter activity and a lengthened clock period. The findings suggest a mechanism by which metabolic factors influence circadian rhythms through AMPK and NAD+ availability.
Abstract
The circadian clock allows living systems to anticipate environmental daily variations by scheduling cellular mechanisms according to the time of the day. The reciprocal interplay between metabolism and circadian clock is essential for energy homeostasis, and the disruption of metabolic clock inputs contributes to dysfunction of clocks in peripheral tissues. However, the molecular mechanisms through which the feeding/fasting cycle entrains peripheral clocks remain incompletely understood. Here, we investigate experimentally and theoretically the role of AMP-dependent kinase (AMPK), a key fasting sensor, in metabolic regulation of the circadian clock. Using a luciferase reporter driven by the Bmal1 promoter to monitor molecular clock activity in human U2OS cells, and thanks to a signal processing method based on the Hilbert transform to retrieve the instantaneous phase and period of circadian signals, we show that pharmacological activation of AMPK by AICAR significantly elevates Bmal1 promoter activity and markedly lengthens the clock period in a dose-dependent manner. Conversely, the clock period is shortened by the pharmacological inhibition by SR18292 of Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1-alpha). Importantly, the action of AICAR is abolished by FK866, a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, demonstrating that AMPK-dependent modulation of Bmal1 promoter activity requires NAD+ availability. These experimental results are recapitulated by numerical simulations of our previously published mathematical model, where PGC-1-alpha plays a key role in mediating AMPK activity to the clock. Moreover, the comparison of experimentally measured and theoretically predicted phase response curves, following AMPK activation at different circadian phases, provides an additional validation of the model. By integrating biological experiments with mathematical modeling, our results identify a key mechanism through which metabolic factors can entrain and regulate the circadian clock via NAD+-dependent AMPK-mediated regulation of Bmal1 promoter activity.
Background
This paper addresses the interaction between metabolism and the circadian clock, focusing on how metabolic signals influence circadian rhythms. Previous research has established that disruptions in metabolic cycles can affect clock function, but the underlying mechanisms remain unclear. Understanding these mechanisms is essential for elucidating how metabolic factors can regulate circadian rhythms.
Methods
The study utilized human U2OS cells to monitor Bmal1 promoter activity through a luciferase reporter. A pharmacological approach was employed, activating AMPK with AICAR and inhibiting PGC-1-alpha with SR18292. The experimental design included numerical simulations to validate the findings.
Results
AICAR activation of AMPK resulted in significant elevation of Bmal1 promoter activity and lengthened the clock period. The study also demonstrated that the effects of AICAR are dependent on NAD+ availability, as shown by the abolition of effects in the presence of FK866.
Interpretation
The findings contribute to the understanding of how AMPK influences circadian rhythms via Bmal1 regulation. While the results are statistically significant, the clinical implications are uncertain due to the in vitro nature of the study and lack of detailed dosage information. The reliance on a single cell line and absence of in vivo validation are notable confounds.
Key findings
- AICAR significantly elevates Bmal1 promoter activity in a dose-dependent manner.
- Pharmacological activation of AMPK by AICAR markedly lengthens the clock period.
- The action of AICAR is abolished by FK866, indicating the requirement of NAD+ availability.
Limitations
- In vitro study using human U2OS cells.
- Specific doses of AICAR not reported.
- No in vivo data to support findings.
- Potential confounding from using a single cell line.