More than the clock: distinct regulation of muscle function and metabolism by PER2 and RORα.

Circadian rhythms, governed by the dominant central clock, in addition to various peripheral clocks, regulate almost all biological processes, including sleep-wake cycles, hormone secretion and metabolism. In certain contexts, the regulation and function of the peripheral oscillations can be decoupled from the central clock. However, the specific mechanisms underlying muscle-intrinsic clock-dependent modulation of muscle function and metabolism remain unclear. We investigated the outcome of perturbations of the primary and secondary feedback loops of the molecular clock in skeletal muscle by specific gene ablation of Period circadian regulator 2 (Per2) and RAR-related orphan receptor alpha (Rorα), respectively. In both models, a dampening of core clock gene oscillation was observed, while the phase was preserved. Moreover, both loops seem to be involved in the homeostasis of amine groups. Highly divergent outcomes were seen for overall muscle gene expression, primarily affecting circadian rhythmicity in the PER2 knockouts and non-oscillating genes in the RORα knockouts, leading to distinct outcomes in terms of metabolome and phenotype. These results highlight the entanglement of the molecular clock and muscle plasticity and allude to specific functions of different clock components, i.e. the primary and secondary feedback loops, in this context. The reciprocal interaction between muscle contractility and circadian clocks might therefore be instrumental to determining a finely tuned adaptation of muscle tissue to perturbations in health and disease. KEY POINTS: Specific perturbations of the primary and secondary feedback loop of the molecular clock result in specific outcomes on muscle metabolism and function. Ablation of Per2 (primary loop) or Rorα (secondary loop) blunts the amplitude of core clock genes, in absence of a shift in phase. Perturbation of the primary feedback loop by deletion of PER2 primarily affects muscle gene oscillation. Knockout of RORα and the ensuing modulation of the secondary loop results in the aberrant expression of a large number of non-clock genes and proteins. The deletion of PER2 and RORα affects muscle metabolism and contractile function in a circadian manner, highlighting the central role of the molecular clock in modulating muscle plasticity.

Transcriptomic, proteomic and phosphoproteomic underpinnings of daily exercise performance and zeitgeber activity of training in mouse muscle.

KEY POINTS: Maximal endurance performance is greater in the early daytime. Timed exercise differentially alters the muscle transcriptome and (phospho)-proteome. Early daytime exercise triggers energy provisioning and tissue regeneration. Early night-time exercise activates stress-related and catabolic pathways. Scheduled training has limited effects on the muscle and liver circadian clocks. ABSTRACT: Timed physical activity might potentiate the health benefits of training. The underlying signalling events triggered by exercise at different times of day are, however, poorly understood. Here, we found that time-dependent variations in maximal treadmill exercise capacity of naïve mice were associated with energy stores, mostly hepatic glycogen levels. Importantly, running at different times of day resulted in a vastly different activation of signalling pathways, e.g. related to stress response, vesicular trafficking, repair and regeneration. Second, voluntary wheel running at the opposite phase of the dark, feeding period surprisingly revealed a minimal zeitgeber (i.e. phase-shifting) effect of training on the muscle clock. This integrated study provides important insights into the circadian regulation of endurance performance and the control of the circadian clock by exercise. In future studies, these results could contribute to better understanding circadian aspects of training design in athletes and the application of chrono-exercise-based interventions in patients.

Circadian clock-dependent and -independent posttranscriptional regulation underlies temporal mRNA accumulation in mouse liver.

The mammalian circadian clock coordinates physiology with environmental cycles through the regulation of daily oscillations of gene expression. Thousands of transcripts exhibit rhythmic accumulations across mouse tissues, as determined by the balance of their synthesis and degradation. While diurnally rhythmic transcription regulation is well studied and often thought to be the main factor generating rhythmic mRNA accumulation, the extent of rhythmic posttranscriptional regulation is debated, and the kinetic parameters (e.g., half-lives), as well as the underlying regulators (e.g., mRNA-binding proteins) are relatively unexplored. Here, we developed a quantitative model for cyclic accumulations of pre-mRNA and mRNA from total RNA-seq data, and applied it to mouse liver. This allowed us to identify that about 20% of mRNA rhythms were driven by rhythmic mRNA degradation, and another 15% of mRNAs regulated by both rhythmic transcription and mRNA degradation. The method could also estimate mRNA half-lives and processing times in intact mouse liver. We then showed that, depending on mRNA half-life, rhythmic mRNA degradation can either amplify or tune phases of mRNA rhythms. By comparing mRNA rhythms in wild-type and Bmal1-/- animals, we found that the rhythmic degradation of many transcripts did not depend on a functional BMAL1. Interestingly clock-dependent and -independent degradation rhythms peaked at distinct times of day. We further predicted mRNA-binding proteins (mRBPs) that were implicated in the posttranscriptional regulation of mRNAs, either through stabilizing or destabilizing activities. Together, our results demonstrate how posttranscriptional regulation temporally shapes rhythmic mRNA accumulation in mouse liver.

Principles for circadian orchestration of metabolic pathways.

Circadian rhythms govern multiple aspects of animal metabolism. Transcriptome-, proteome- and metabolome-wide measurements have revealed widespread circadian rhythms in metabolism governed by a cellular genetic oscillator, the circadian core clock. However, it remains unclear if and under which conditions transcriptional rhythms cause rhythms in particular metabolites and metabolic fluxes. Here, we analyzed the circadian orchestration of metabolic pathways by direct measurement of enzyme activities, analysis of transcriptome data, and developing a theoretical method called circadian response analysis. Contrary to a common assumption, we found that pronounced rhythms in metabolic pathways are often favored by separation rather than alignment in the times of peak activity of key enzymes. This property holds true for a set of metabolic pathway motifs (e.g., linear chains and branching points) and also under the conditions of fast kinetics typical for metabolic reactions. By circadian response analysis of pathway motifs, we determined exact timing separation constraints on rhythmic enzyme activities that allow for substantial rhythms in pathway flux and metabolite concentrations. Direct measurements of circadian enzyme activities in mouse skeletal muscle confirmed that such timing separation occurs in vivo.

Diurnal variation in egg excretion by Heterakis gallinarum.

Periodicity in nematode egg excretion may be of evolutionary origin as it can favour dispersal of the eggs in the environment. We investigated whether egg excretion by Heterakis gallinarum shows a repeatable pattern of periodicity. The faecal egg concentration and total number of eggs excreted within 4-h intervals were significantly affected by the sampling time within 1 day, but remained unaffected by the sampling day or interaction effects. By contrast, the total number of eggs excreted within 24 h did not differ among the 4 days of the study, collectively indicating repeatable egg excretion patterns. Both host feces and parasite egg excretion increased from night to late afternoon, followed by a decrease in the evening, resulting in higher egg excretion during daytime than the dark period. Feces excretion and worm fecundity showed overlapping diurnal rhythms with similarly timed phases, suggesting the existence of synchronicity between the host feces and nematode egg excretion patterns. We conclude that egg excretion by H. gallinarum is synchronized with host feces excretion and is higher during the daytime than during the dark period. This overlaps with the maximum activity of the day-active host and allows a maximal dispersal of the eggs in the environment.

Linking Core Promoter Classes to Circadian Transcription.

Circadian rhythms in transcription are generated by rhythmic abundances and DNA binding activities of transcription factors. Propagation of rhythms to transcriptional initiation involves the core promoter, its chromatin state, and the basal transcription machinery. Here, I characterize core promoters and chromatin states of genes transcribed in a circadian manner in mouse liver and in Drosophila. It is shown that the core promoter is a critical determinant of circadian mRNA expression in both species. A distinct core promoter class, strong circadian promoters (SCPs), is identified in mouse liver but not Drosophila. SCPs are defined by specific core promoter features, and are shown to drive circadian transcriptional activities with both high averages and high amplitudes. Data analysis and mathematical modeling further provided evidence for rhythmic regulation of both polymerase II recruitment and pause release at SCPs. The analysis provides a comprehensive and systematic view of core promoters and their link to circadian mRNA expression in mouse and Drosophila, and thus reveals a crucial role for the core promoter in regulated, dynamic transcription.

Differential rhythmicity: detecting altered rhythmicity in biological data.

MOTIVATION: Biological rhythms, such as rhythms in gene expression controlled by the cell cycle or the circadian clock, are important in cell physiology. A common type of experiment compares rhythmicity in tissues or cells either kept under different conditions or having different genotypes. Such investigations provide insights into underlying mechanisms as well as functions of rhythms. RESULTS: We present and benchmark a set of statistical and computational methods for this type of analysis, here termed differential rhythmicity analysis. The methods detect alterations in rhythm amplitude, phase and signal to noise ratio in one set of measurements compared to another. Using these methods, we compared circadian rhythms in liver mRNA expression in mice held under two different lighting conditions: constant darkness and light-dark cycles, respectively. This analysis revealed widespread and reproducible amplitude increases in mice kept in light-dark cycles. Further analysis of the subset of differentially rhythmic transcripts implied the immune system in mediating ambient light-dark cycles to rhythmic transcriptional activities. The methods are suitable for genome- or proteome-wide studies, and provide rigorous P values against well-defined null hypotheses. AVAILABILITY AND IMPLEMENTATION: The methods were implemented as the accompanying R software package DODR, available on CRAN. CONTACT: pal-olof.westermark@charite.de SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Circadian mRNA expression: insights from modeling and transcriptomics.

Circadian clocks synchronize organisms to the 24 h rhythms of the environment. These clocks persist under constant conditions, have their origin at the cellular level, and produce an output of rhythmic mRNA expression affecting thousands of transcripts in many mammalian cell types. Here, we review the charting of circadian output rhythms in mRNA expression, focusing on mammals. We emphasize the challenges in statistics, interpretation, and quantitative descriptions that such investigations have faced and continue to face, and outline remaining outstanding questions.

Dynamics of fat cell turnover in humans.

Obesity is increasing in an epidemic manner in most countries and constitutes a public health problem by enhancing the risk for cardiovascular disease and metabolic disorders such as type 2 diabetes. Owing to the increase in obesity, life expectancy may start to decrease in developed countries for the first time in recent history. The factors determining fat mass in adult humans are not fully understood, but increased lipid storage in already developed fat cells (adipocytes) is thought to be most important. Here we show that adipocyte number is a major determinant for the fat mass in adults. However, the number of fat cells stays constant in adulthood in lean and obese individuals, even after marked weight loss, indicating that the number of adipocytes is set during childhood and adolescence. To establish the dynamics within the stable population of adipocytes in adults, we have measured adipocyte turnover by analysing the integration of 14C derived from nuclear bomb tests in genomic DNA. Approximately 10% of fat cells are renewed annually at all adult ages and levels of body mass index. Neither adipocyte death nor generation rate is altered in early onset obesity, suggesting a tight regulation of fat cell number in this condition during adulthood. The high turnover of adipocytes establishes a new therapeutic target for pharmacological intervention in obesity.