Circadian regulation of cellular physiology

Abstract

The circadian clock synchronizes behavioral and physiological processes on a daily basis in anticipation of the light-dark cycle. In mammals, molecular clocks are present in both the central pacemaker neurons and in nearly all peripheral tissues. Clock transcription factors in metabolic tissues coordinate metabolic fuel utilization and storage with alternating periods of feeding and fasting corresponding to the rest-activity cycle. In vitro and in vivo biochemical approaches have led to the discovery of mechanisms underlying the interplay between the molecular clock and the metabolic networks. For example, recent studies have demonstrated that the circadian clock controls rhythmic synthesis of the cofactor nicotinamide adenine dinucleotide (NAD(+)) and activity of NAD(+)-dependent sirtuin deacetylase enzymes to regulate mitochondrial function across the circadian cycle. In this chapter, we review current state-of-the-art methods to analyze circadian cycles in mitochondrial bioenergetics, glycolysis, and nucleotide metabolism in both cell-based and animal models.

Keywords: Circadian; Energetics; Metabolism; Mitochondria; NAD; Respiration; Sirtuin.

Figure 1

Robust in vitro circadian oscillations in synchronized C2C12 cells. Real-time bioluminescence recordings from PER2:LUC-expressing C2C12 myotubes. Bioluminescence was monitored for 3 days in a Lumicycler apparatus following synchronization of the cells with 50% horse serum.

Figure 2

Serial synchronization for 48 h with single collection time point for C2C12 cells. A staggered synchronization every 4 h for a total of 48 h enables a single collection time point, which is necessary for assays that require simultaneous comparison of samples in live cells.

Figure 3

Model of fuel substrates and pharmacological mitochondrial respiratory modulators used in the in vitro bioenergetics measurements. Using a Seahorse Biosciences XF96 Bioanalyzer, small volumes of intact and permeabilized cells can be analyzed for both mitochondrial respiration (i.e., oxygen consumption rate—OCR) and lactate production via anaerobic glycolysis (i.e., extracellular medium acidification rate—ECAR). To compare utilization of fuel substrates, several oxidative substrates can be added to permeabilized cells, including acyl-carnitines to measure FAO, pyruvate/malate to measure flux from glucose into the mitochondria, glutamate/malate to measure flux from glutamine, and succinate plus rotenone (a complex I inhibitor) to measure respiratory chain from complex II. Respiratory function can also be analyzed in detail using pharmacological modulators of the electron transport chain, including oligomycin (inhibits ATP synthase and measures proton leak), FCCP (dissipates inner membrane proton gradient and measures maximal respiratory capacity), and antimycin A (inhibits complex III and measures nonrespiratory OCR).

Figure 4

Experimental timeline for in vivo 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 CT0). 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.