Daily running enhances molecular and physiological circadian rhythms in skeletal muscle.

OBJECTIVE: Exercise is a critical component of a healthy lifestyle and a key strategy for the prevention and management of metabolic disease. Identifying molecular mechanisms underlying adaptation in response to chronic physical activity is of critical interest in metabolic physiology. Circadian rhythms broadly modulate metabolism, including muscle substrate utilization and exercise capacity. Here, we define the molecular and physiological changes induced across the daily cycle by voluntary low intensity daily exercise. METHODS: Wildtype C57BL6/J male and female mice were housed with or without access to a running wheel for six weeks. Maximum running speed was measured at four different zeitgeber times (ZTs, hours after lights on) using either electrical or manual stimulation to motivate continued running on a motorized treadmill. RNA isolated from plantaris muscles at six ZTs was sequenced to establish the impact of daily activity on genome-wide transcription. Patterns of gene expression were analyzed using Gene Set Enrichment Analysis (GSEA) and Detection of Differential Rhythmicity (DODR). Blood glucose, lactate, and ketones, and muscle and liver glycogen were measured before and after exercise. RESULTS: We demonstrate that the use of mild electrical shocks to motivate running negatively impacts maximum running speed in mice, and describe a manual method to motivate running in rodent exercise studies. Using this method, we show that time of day influences the increase in exercise capacity afforded by six weeks of voluntary wheel running: when maximum running speed is measured at the beginning of the nighttime active period in mice, there is no measurable benefit from a history of daily voluntary running, while maximum increase in performance occurs at the end of the night. We show that daily voluntary exercise dramatically remodels the murine muscle circadian transcriptome. Finally, we describe daily rhythms in carbohydrate metabolism associated with the time-dependent response to moderate daily exercise in mice. CONCLUSIONS: Collectively, these data indicate that chronic nighttime physical activity dramatically remodels daily rhythms of murine muscle gene expression, which in turn support daily fluctuations in exercise performance.

Cryptochromes Suppress HIF1α in Muscles.

Muscles preferentially utilize glycolytic or oxidative metabolism depending on the intensity of physical activity. Transcripts required for carbohydrate and lipid metabolism undergo circadian oscillations of expression in muscles, and both exercise capacity and the metabolic response to exercise are influenced by time of day. The circadian repressors CRY1 and CRY2 repress peroxisome proliferator-activated receptor delta (PPARδ), a major driver of oxidative metabolism and exercise endurance. CRY-deficient mice exhibit enhanced PPARδ activation and greater maximum speed when running on a treadmill but no increase in exercise endurance. Here we demonstrate that CRYs limit hypoxia-responsive transcription via repression of HIF1α-BMAL1 heterodimers. Furthermore, CRY2 appeared to be more effective than CRY1 in the reduction of HIF1α protein steady-state levels in primary myotubes and quadriceps in vivo. Finally, CRY-deficient myotubes exhibit metabolic alterations consistent with cryptochrome-dependent suppression of HIF1α, which likely contributes to circadian modulation of muscle metabolism.

The Liver Circadian Clock Modulates Biochemical and Physiological Responses to Metformin.

Metformin is widely used in the treatment of type 2 diabetes to lower blood glucose. Although metformin is a relatively safe and effective drug, its clinical efficacy is variable and under certain circumstances it may contribute to life-threatening lactic acidosis. Thus, additional understanding of metformin pharmacokinetics and pharmacodynamics could provide important information regarding therapeutic use of this widely prescribed drug. Here we report a significant effect of time of day on acute blood glucose reduction in response to metformin administration and on blood lactate levels in healthy mice. Furthermore, we demonstrate that while metformin transport into hepatocytes is unaltered by time of day, the kinetics of metformin-induced activation of AMP-activated protein kinase (AMPK) in the liver are remarkably altered with circadian time. Liver-specific ablation of Bmal1 expression alters metformin induction of AMPK and blood glucose response but does not completely abolish time of day differences. Together, these data demonstrate that circadian rhythms affect the biological responses to metformin in a complex manner.

Circadian repressors CRY1 and CRY2 broadly interact with nuclear receptors and modulate transcriptional activity.

Nuclear hormone receptors (NRs) regulate physiology by sensing lipophilic ligands and adapting cellular transcription appropriately. A growing understanding of the impact of circadian clocks on mammalian transcription has sparked interest in the interregulation of transcriptional programs. Mammalian clocks are based on a transcriptional feedback loop featuring the transcriptional activators circadian locomotor output cycles kaput (CLOCK) and brain and muscle ARNT-like 1 (BMAL1), and transcriptional repressors cryptochrome (CRY) and period (PER). CRY1 and CRY2 bind independently of other core clock factors to many genomic sites, which are enriched for NR recognition motifs. Here we report that CRY1/2 serve as corepressors for many NRs, indicating a new facet of circadian control of NR-mediated regulation of metabolism and physiology, and specifically contribute to diurnal modulation of drug metabolism.