Circadian measurements of sirtuin biology

Abstract

Many of our behavioral and physiological processes display daily oscillations that are under the control of the circadian clock. The core molecular clock network is present in both the brain and peripheral tissues and is composed of a complex series of interlocking transcriptional/translational feedback loops that oscillate with a periodicity of ~24 h. Recent evidence has implicated NAD(+) biosynthesis and the sirtuin family of NAD(+)-dependent protein deacetylases as part of a novel feedback loop within the core clock network, findings which underscore the importance of taking circadian timing into consideration when designing and interpreting metabolic studies, particularly in regard to sirtuin biology. Thus, this chapter introduces both in vivo and in vitro circadian methods to analyze various sirtuin-related endpoints across the light-dark cycle and discusses the transcriptional, biochemical, and physiological outputs of the clock.

Fig. 1

Molecular overview of core clock network. The core molecular clock network consists of a series of positive and negative transcription/translation feedback loops. The positive limb consists of CLOCK/BMAL1, which heterodimerizes and transactivates expression of downstream target genes containing an E-box in their promoters, including the Period, Cryptochrome, Ror, Rev-erb, and Nampt genes. The PER and CRY proteins multimerize and inhibit the activity of CLOCK/BMAL1, until they are degraded following CKI- and FBLX3-mediated phosphorylation and ubiquitination, respectively. Rhythmic generation of Nampt results in oscillations in NAD⁺ and SIRT activity, which feeds back to regulate the positive limb of the clock

Fig. 2

In vivo experimental timeline for 48 h tissue collection in constant darkness. Mice are released into DD at ZT12 in order to coordinate with the start of the normal dark period. Food is removed from the first set of mice at CT6, which is 18 h prior to the first tissue collection (indicated by CTO). The fasting and tissue collection is then repeated every 4 h for 44 h in order to get a full set of samples to be analyzed for rhythms of the process of interest

Fig. 3

Example of double-plotted actogram of locomotor activity. Wheel turns are recorded in 5-min blocks and plotted as histograms along a horizontal line representing the time of day. The actogram is double plotted, meaning that 2 consecutive days are positioned next to each other both horizontally and vertically. Lighting conditions are indicated to the right. The arrow denotes the switch from 12:12 LD to constant darkness (DD) on day 10

Fig. 4

In vitro experimental timelines for synchronization and 48 h collection time points in cells. Three different experimental setups for synchronizing cells in culture are presented. Method 1 entails synchronization of all cells at one time point, followed by collection of samples every 4 h over the course of 48 h. Method 2 entails synchronization of the cells every 4 h, enabling a single collection time point (which may be necessary if a particular assay requires all samples be run at the same time). Finally, Method 3 (described in text) outlines a convenient approach whereby cells are synchronized at four time points over 36 h and then collected at three time points on the final day of the experiment such that no overnight stays are required

Fig. 5

Monitoring PER2-LUC oscillations in vitro. MEFs can be isolated from either PER2-LUC mice or can be infected with PER2-LUC lentivirus. Cells are then synchronized by the addition of forskolin directly to the media, and bioluminescence can be monitored for the remainder of the experiment in a Lumicycler machine. Each plate should display ∼24 h rhythms of luminescence beginning from 12 to 24 h after forskolin treatment. An example trace is shown