SUMMARY Cellular metabolite balance and mitochondrial function are under circadian control, but the pathways connecting the molecular clock to these functions are unclear. Peroxisome proliferator activated receptor delta (PPARδ) enables preferential utilization of lipids as fuel during exercise and is a major driver of exercise endurance. We show here that the circadian repressors CRY1 and CRY2 function as co-repressors for PPARδ. Cry1−/−;Cry2−/− myotubes and muscles exhibit elevated expression of PPARδ target genes, particularly in the context of exercise. Notably, CRY1/2 seem to repress a distinct subset of PPARδ target genes in muscle compared to the co-repressor NCOR1. In vivo, genetic disruption of Cry1 and Cry2 enhances sprint exercise performance in mice. Collectively, our data demonstrate that CRY1 and CRY2 modulate exercise physiology by altering the activity of several transcription factors, including CLOCK/BMAL1 and PPARδ and thereby alter energy storage and substrate selection for energy production.

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.

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Metformin is widely used in the treatment of type 2 diabetes to lower blood glucose. Though it is a relatively safe and effective drug, 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 usage 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 impact the biological responses to metformin in a complex manner.

Research over the past century indicates that the daily timing of physical activity impacts both immediate performance and long-term training efficacy. Recently, several molecular connections between circadian clocks and exercise physiology have been identified. Circadian clocks are protein-based oscillators that enable anticipation of daily environmental cycles. Cell-autonomous clocks exist in almost all cells of the body and their timing is set by a variety of internal and external signals, including hormones and dietary intake. Improved understanding of the relationship between molecular clocks and exercise will benefit professional athletes and public health guidelines for the general population. Here, we discuss the role of circadian clocks in exercise, exploring time-of-day effects and proposed molecular and physiological mechanisms.

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BackgroundNitric oxide synthase uncoupling occurs under conditions of oxidative stress modifying the enzyme's function so it generates superoxide rather than nitric oxide. Nitric oxide synthase uncoupling occurs with chronic pressure overload, and both are ameliorated by exogenous tetrahydrobiopterin (BH4)—a cofactor required for normal nitric oxide synthase function—supporting a pathophysiological link. Genetically augmenting BH4 synthesis in endothelial cells fails to replicate this benefit, indicating that other cell types dominate the effects of exogenous BH4 administration. We tested whether the primary cellular target of BH4 is the cardiomyocyte or whether other novel mechanisms are invoked.Methods and ResultsMice with cardiomyocyte‐specific overexpression of GTP cyclohydrolase 1 (mGCH1) and wild‐type littermates underwent transverse aortic constriction. The mGCH1 mice had markedly increased myocardial BH4 and, unlike wild type, maintained nitric oxide synthase coupling after transverse aortic constriction; however, the transverse aortic constriction–induced abnormalities in cardiac morphology and function were similar in both groups. In contrast, exogenous BH4 supplementation improved transverse aortic constricted hearts in both groups, suppressed multiple inflammatory cytokines, and attenuated infiltration of inflammatory macrophages into the heart early after transverse aortic constriction.Conclusions BH4 protection against adverse remodeling in hypertrophic cardiac disease is not driven by its prevention of myocardial nitric oxide synthase uncoupling, as presumed previously. Instead, benefits from exogenous BH4 are mediated by a protective effect coupled to suppression of inflammatory pathways and myocardial macrophage infiltration.

Rationale: In diabetic patients, heart failure with predominant left ventricular (LV) diastolic dysfunction is a common complication for which there is no effective treatment. Oxidation of the nitric oxide synthase (NOS) co-factor tetrahydrobiopterin (BH4) and dysfunctional NOS activity have been implicated in the pathogenesis of the diabetic vascular and cardiomyopathic phenotype. Objective: Using mice models and human myocardial samples, we evaluated whether and by which mechanism increasing myocardial BH4 availability prevented or reversed LV dysfunction induced by diabetes. Methods and Results: In contrast to the vascular endothelium, BH4 levels, superoxide production and NOS activity (by liquid chromatography) did not differ in the LV myocardium of diabetic mice or in atrial tissue from diabetic patients. Nevertheless, the impairment in both cardiomyocyte relaxation and [Ca2+]i decay and in vivo LV function (echocardiography and tissue Doppler) that developed in wild type mice (WT) 12 weeks post-DM induction (streptozotocin, 42-45mg/kg) was prevented in mice with elevated myocardial BH4 content secondary to overexpression of GTP-cyclohydrolase 1 (mGCH1-Tg) and reversed in WT mice receiving oral BH4 supplementation from the 12th to the 18th week after DM induction. The protective effect of BH4 was abolished by CRISPR/Cas9-mediated knockout of neuronal NOS (nNOS) in mGCH1-Tg. In HEK cells, S-nitrosoglutathione led to a PKG-dependent increase in plasmalemmal density of the insulin-independent glucose transporter, GLUT-1. In cardiomyocytes, mGCH1 overexpression induced a NO/sGC/PKG-dependent increase in glucose uptake via GLUT-1, which was instrumental in preserving mitochondrial creatine kinase activity, oxygen consumption rate, LV energetics (by 31P MRS) and myocardial function. Conclusions: We uncovered a novel mechanism whereby myocardial BH4 prevents and reverses LV diastolic and systolic dysfunction associated with diabetes via a nNOS-mediated increase in non-insulin dependent myocardial glucose uptake and utilization. These findings highlight the potential of GCH1/BH4-based therapeutics in human diabetic cardiomyopathy.