Tight regulation of a nuclear HAPSTR1-HUWE1 pathway essential for mammalian life.

The recently discovered HAPSTR1 protein broadly oversees cellular stress responses. This function requires HUWE1, a ubiquitin ligase that paradoxically marks HAPSTR1 for degradation, but much about this pathway remains unclear. Here, leveraging multiplexed proteomics, we find that HAPSTR1 enables nuclear localization of HUWE1 with implications for nuclear protein quality control. We show that HAPSTR1 is tightly regulated and identify ubiquitin ligase TRIP12 and deubiquitinase USP7 as upstream regulators titrating HAPSTR1 stability. Finally, we generate conditional Hapstr1 knockout mice, finding that Hapstr1-null mice are perinatal lethal, adult mice depleted of Hapstr1 have reduced fitness, and primary cells explanted from Hapstr1-null animals falter in culture coincident with HUWE1 mislocalization and broadly remodeled signaling. Notably, although HAPSTR1 potently suppresses p53, we find that Hapstr1 is essential for life even in mice lacking p53. Altogether, we identify novel components and functional insights into the conserved HAPSTR1-HUWE1 pathway and demonstrate its requirement for mammalian life.

Impact of circadian time of dosing on cardiomyocyte-autonomous effects of glucocorticoids.

OBJECTIVE: Mitochondrial capacity is critical to adapt the high energy demand of the heart to circadian oscillations and diseased states. Glucocorticoids regulate the circadian cycle of energy metabolism, but little is known about how circadian timing of exogenous glucocorticoid dosing directly regulates heart metabolism through cardiomyocyte-autonomous mechanisms. While chronic once-daily intake of glucocorticoids promotes metabolic stress and heart failure, we recently discovered that intermittent once-weekly dosing of exogenous glucocorticoids promoted muscle metabolism in normal and obese skeletal muscle. However, the effects of glucocorticoid intermittence on heart metabolism and heart failure remain unknown. Here we investigated the extent to which circadian time of dosing regulates the effects of the glucocorticoid prednisone in heart metabolism and function in conditions of single pulse or chronic intermittent dosing. METHODS AND RESULTS: In WT mice, we found that prednisone improved cardiac content of NAD+ and ATP with light-phase dosing (ZT0), while the effects were blocked by dark-phase dosing (ZT12). The drug effects on mitochondrial function were cardiomyocyte-autonomous, as shown by inducible cardiomyocyte-restricted glucocorticoid receptor (GR) ablation, and depended on an intact cardiomyocyte clock, as shown by inducible cardiomyocyte-restricted ablation of Brain and Muscle ARNT-like 1 (BMAL1). Conjugating time-of-dosing with chronic intermittence, we found that once-weekly prednisone improved metabolism and function in heart after myocardial injury dependent on circadian time of intake, i.e. with light-phase but not dark-phase dosing. CONCLUSIONS: Our study identifies cardiac-autonomous mechanisms through which circadian-specific intermittent dosing reconverts glucocorticoid drugs to metabolic boosters for the heart.

BMAL1 drives muscle repair through control of hypoxic NAD+ regeneration in satellite cells.

The process of tissue regeneration occurs in a developmentally timed manner, yet the role of circadian timing is not understood. Here, we identify a role for the adult muscle stem cell (MuSC)-autonomous clock in the control of muscle regeneration following acute ischemic injury. We observed greater muscle repair capacity following injury during the active/wake period as compared with the inactive/rest period in mice, and loss of Bmal1 within MuSCs leads to impaired muscle regeneration. We demonstrate that Bmal1 loss in MuSCs leads to reduced activated MuSC number at day 3 postinjury, indicating a failure to properly expand the myogenic precursor pool. In cultured primary myoblasts, we observed that loss of Bmal1 impairs cell proliferation in hypoxia (a condition that occurs in the first 1-3 d following tissue injury in vivo), as well as subsequent myofiber differentiation. Loss of Bmal1 in both cultured myoblasts and in vivo activated MuSCs leads to reduced glycolysis and premature activation of prodifferentiation gene transcription and epigenetic remodeling. Finally, hypoxic cell proliferation and myofiber formation in Bmal1-deficient myoblasts are restored by increasing cytosolic NAD+ Together, we identify the MuSC clock as a pivotal regulator of oxygen-dependent myoblast cell fate and muscle repair through the control of the NAD+-driven response to injury.

Muscle mitochondrial remodeling by intermittent glucocorticoid drugs requires an intact circadian clock and muscle PGC1α.

Exogenous glucocorticoids interact with the circadian clock, but little attention is paid to the timing of intake. We recently found that intermittent once-weekly prednisone improved nutrient oxidation in dystrophic muscle. Here, we investigated whether dosage time affected prednisone effects on muscle bioenergetics. In mice treated with once-weekly prednisone, drug dosing in the light-phase promoted nicotinamide adenine dinucleotide (NAD+) levels and mitochondrial function in wild-type muscle, while this response was lost with dark-phase dosing. These effects depended on a normal circadian clock since they were disrupted in muscle from [Brain and muscle Arnt-like protein-1 (Bmal1)]-knockout mice. The light-phase prednisone pulse promoted BMAL1-dependent glucocorticoid receptor recruitment on noncanonical targets, including Nampt and Ppargc1a [peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α)]. In mice with muscle-restricted inducible PGC1α ablation, bioenergetic stimulation by light-phase prednisone required PGC1α. These results demonstrate that glucocorticoid "chronopharmacology" for muscle bioenergetics requires an intact clock and muscle PGC1α activity.

NADH inhibition of SIRT1 links energy state to transcription during time-restricted feeding.

In mammals, circadian rhythms are entrained to the light cycle and drive daily oscillations in levels of NAD+, a cosubstrate of the class III histone deacetylase sirtuin 1 (SIRT1) that associates with clock transcription factors. Although NAD+ also participates in redox reactions, the extent to which NAD(H) couples nutrient state with circadian transcriptional cycles remains unknown. Here we show that nocturnal animals subjected to time-restricted feeding of a calorie-restricted diet (TRF-CR) only during night-time display reduced body temperature and elevated hepatic NADH during daytime. Genetic uncoupling of nutrient state from NADH redox state through transduction of the water-forming NADH oxidase from Lactobacillus brevis (LbNOX) increases daytime body temperature and blood and liver acyl-carnitines. LbNOX expression in TRF-CR mice induces oxidative gene networks controlled by brain and muscle Arnt-like protein 1 (BMAL1) and peroxisome proliferator-activated receptor alpha (PPARα) and suppresses amino acid catabolic pathways. Enzymatic analyses reveal that NADH inhibits SIRT1 in vitro, corresponding with reduced deacetylation of SIRT1 substrates during TRF-CR in vivo. Remarkably, Sirt1 liver nullizygous animals subjected to TRF-CR display persistent hypothermia even when NADH is oxidized by LbNOX. Our findings reveal that the hepatic NADH cycle links nutrient state to whole-body energetics through the rhythmic regulation of SIRT1.

Associations of Poly (ADP-Ribose) Polymerase1 abundance in calf skeletal muscle with walking performance in peripheral artery disease.

OBJECTIVE: This study investigated associations of markers of oxidative stress and mitochondrial function in calf muscle biopsies with walking performance in people with and without lower extremity peripheral artery disease (PAD). METHODS: Participants with PAD (ankle-brachial index (ABI) <0.90) and without PAD (ABI: 0.90-1.50) underwent calf muscle biopsy and measurement of 6-min walk and four-meter walking velocity. PARP1 (Poly (ADP-Ribose) Polymerase 1), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), silent information regulator 1 (SIRT1) and 4-hydroxynonenal (4HNE) expression were measured in calf muscle using western blot. RESULTS: Among 15 participants with PAD mean age: 66.8 years (standard deviation (SD): 6.4) and six without PAD (age: 64.4 years, SD: 5.9), mean PARP1-abundance in calf muscle was 1.16 ± 0.92 AU and 0.96 ± 0.38 AU, respectively (P = 0.61). Among participants with PAD after adjustment with ABI, a greater abundance of PARP1 was associated with poorer 6-min walking distance (r = -0.65, P = 0.01), usual-paced 4-m walking velocity (r = -0.73, P = 0.003) and slower fast-paced four-meter walking velocity (r = -0.51, P = 0.07). Among participants with PAD, ABI was not associated with PARP1 abundance in calf muscle (r = 0.02, P = 0.93). Among participants without PAD, skeletal muscle PARP1 abundance was not significantly associated with 6-min walk distance (r = -0.58; P = 0.22), usual-paced walking velocity (r = -0.26; P = 0.62), or fast-paced walking velocity (r = -0.21; P = 0.69), perhaps due to lack of statistical power. There were no associations of remaining calf muscle measures with walking performance. CONCLUSIONS: These findings are consistent with the hypothesis that calf skeletal muscle characteristics are related to walking performance, independently of severity of lower extremity arterial obstruction in people with PAD.

Metabolic Implications of Circadian-HIF Crosstalk.

Research over the past few decades has shed light on the mechanisms underlying the link between circadian disruption and the development of metabolic diseases such as obesity, type 2 diabetes, and cancer. However, how the clock network interacts with tissue-specificnutrient-sensing pathways during conditions of nutrient stress or pathological states remains incompletely understood. Recent work has demonstrated that the circadian clock can 'reprogram' the transcriptome to control distinct sets of genes during altered nutrient conditions, such as high fat diet, aging, and exercise. In this review, I discuss connections between circadian clock transcription factors and the oxygen- and nutrient-responsivehypoxia-inducible factor (HIF) pathway. I highlight recently uncovered mechanistic insights underlying these pathway interactions and address potential implications for the role of circadian disruption in metabolic diseases.

NAD+ Controls Circadian Reprogramming through PER2 Nuclear Translocation to Counter Aging.

Disrupted sleep-wake and molecular circadian rhythms are a feature of aging associated with metabolic disease and reduced levels of NAD+, yet whether changes in nucleotide metabolism control circadian behavioral and genomic rhythms remains unknown. Here, we reveal that supplementation with the NAD+ precursor nicotinamide riboside (NR) markedly reprograms metabolic and stress-response pathways that decline with aging through inhibition of the clock repressor PER2. NR enhances BMAL1 chromatin binding genome-wide through PER2K680 deacetylation, which in turn primes PER2 phosphorylation within a domain that controls nuclear transport and stability and that is mutated in human advanced sleep phase syndrome. In old mice, dampened BMAL1 chromatin binding, transcriptional oscillations, mitochondrial respiration rhythms, and late evening activity are restored by NAD+ repletion to youthful levels with NR. These results reveal effects of NAD+ on metabolism and the circadian system with aging through the spatiotemporal control of the molecular clock.

Pulsed glucocorticoids enhance dystrophic muscle performance through epigenetic-metabolic reprogramming.

In humans, chronic glucocorticoid use is associated with side effects like muscle wasting, obesity, and metabolic syndrome. Intermittent steroid dosing has been proposed in Duchenne Muscular Dystrophy patients to mitigate the side effects seen with daily steroid intake. We evaluated biomarkers from Duchenne Muscular Dystrophy patients, finding that, compared with chronic daily steroid use, weekend steroid use was associated with reduced serum insulin, free fatty acids, and branched chain amino acids, as well as reduction in fat mass despite having similar BMIs. We reasoned that intermittent prednisone administration in dystrophic mice would alter muscle epigenomic signatures, and we identified the coordinated action of the glucocorticoid receptor, KLF15 and MEF2C as mediators of a gene expression program driving metabolic reprogramming and enhanced nutrient utilization. Muscle lacking Klf15 failed to respond to intermittent steroids. Furthermore, coadministration of the histone acetyltransferase inhibitor anacardic acid with steroids in mdx mice eliminated steroid-specific epigenetic marks and abrogated the steroid response. Together, these findings indicate that intermittent, repeated exposure to glucocorticoids promotes performance in dystrophic muscle through an epigenetic program that enhances nutrient utilization.

Cryptochromes-Mediated Inhibition of the CRL4Cop1-Complex Assembly Defines an Evolutionary Conserved Signaling Mechanism.

In plants, cryptochromes are photoreceptors that negatively regulate the ubiquitin ligase CRL4Cop1. In mammals, cryptochromes are core components of the circadian clock and repressors of the glucocorticoid receptor (GR). Moreover, mammalian cryptochromes lost their ability to interact with Cop1, suggesting that they are unable to inhibit CRL4Cop1. Contrary to this assumption, we found that mammalian cryptochromes are also negative regulators of CRL4Cop1, and through this mechanism, they repress the GR transcriptional network both in cultured cells and in the mouse liver. Mechanistically, cryptochromes inactivate Cop1 by interacting with Det1, a subunit of the mammalian CRL4Cop1 complex that is not present in other CRL4s. Through this interaction, the ability of Cop1 to join the CRL4 complex is inhibited; therefore, its substrates accumulate. Thus, the interaction between cryptochromes and Det1 in mammals mirrors the interaction between cryptochromes and Cop1 in planta, pointing to a common ancestor in which the cryptochromes-Cop1 axis originated.

Transcriptional Basis for Rhythmic Control of Hunger and Metabolism within the AgRP Neuron.

The alignment of fasting and feeding with the sleep/wake cycle is coordinated by hypothalamic neurons, though the underlying molecular programs remain incompletely understood. Here, we demonstrate that the clock transcription pathway maximizes eating during wakefulness and glucose production during sleep through autonomous circadian regulation of NPY/AgRP neurons. Tandem profiling of whole-cell and ribosome-bound mRNAs in morning and evening under dynamic fasting and fed conditions identified temporal control of activity-dependent gene repertoires in AgRP neurons central to synaptogenesis, bioenergetics, and neurotransmitter and peptidergic signaling. Synaptic and circadian pathways were specific to whole-cell RNA analyses, while bioenergetic pathways were selectively enriched in the ribosome-bound transcriptome. Finally, we demonstrate that the AgRP clock mediates the transcriptional response to leptin. Our results reveal that time-of-day restriction in transcriptional control of energy-sensing neurons underlies the alignment of hunger and food acquisition with the sleep/wake state.

ER-associated ubiquitin ligase HRD1 programs liver metabolism by targeting multiple metabolic enzymes.

The HMG-CoA reductase degradation protein 1 (HRD1) has been identified as a key enzyme for endoplasmic reticulum-associated degradation of misfolded proteins, but its organ-specific physiological functions remain largely undefined. Here we show that mice with HRD1 deletion specifically in the liver display increased energy expenditure and are resistant to HFD-induced obesity and liver steatosis and insulin resistance. Proteomic analysis identifies a HRD1 interactome, a large portion of which includes metabolic regulators. Loss of HRD1 results in elevated ENTPD5, CPT2, RMND1, and HSD17B4 protein levels and a consequent hyperactivation of both AMPK and AKT pathways. Genome-wide mRNA sequencing revealed that HRD1-deficiency reprograms liver metabolic gene expression profiles, including suppressing genes involved in glycogenesis and lipogenesis and upregulating genes involved in glycolysis and fatty acid oxidation. We propose HRD1 as a liver metabolic regulator and a potential drug target for obesity, fatty liver disease, and insulin resistance associated with the metabolic syndrome.

Circadian Clock Interaction with HIF1α Mediates Oxygenic Metabolism and Anaerobic Glycolysis in Skeletal Muscle.

Circadian clocks are encoded by a transcription-translation feedback loop that aligns energetic processes with the solar cycle. We show that genetic disruption of the clock activator BMAL1 in skeletal myotubes and fibroblasts increased levels of the hypoxia-inducible factor 1α (HIF1α) under hypoxic conditions. Bmal1-/- myotubes displayed reduced anaerobic glycolysis, mitochondrial respiration with glycolytic fuel, and transcription of HIF1α targets Phd3, Vegfa, Mct4, Pk-m, and Ldha, whereas abrogation of the clock repressors CRY1/2 stabilized HIF1α in response to hypoxia. HIF1α bound directly to core clock gene promoters, and, when co-expressed with BMAL1, led to transactivation of PER2-LUC and HRE-LUC reporters. Further, genetic stabilization of HIF1α in Vhl-/- cells altered circadian transcription. Finally, induction of clock and HIF1α target genes in response to strenuous exercise varied according to the time of day in wild-type mice. Collectively, our results reveal bidirectional interactions between circadian and HIF pathways that influence metabolic adaptation to hypoxia.

Circadian rhythms and metabolism: from the brain to the gut and back again.

This paper focuses on the relationship between the circadian system and glucose metabolism. Research across the translational spectrum confirms the importance of the circadian system for glucose metabolism and offers promising clues as to when and why these systems go awry. In particular, basic research has started to clarify the molecular and genetic mechanisms through which the circadian system regulates metabolism. The study of human behavior, especially in the context of psychiatric disorders, such as bipolar disorder and major depression, forces us to see how inextricably linked mental health and metabolic health are. We also emphasize the remarkable opportunities for advancing circadian science through big data and advanced analytics. Advances in circadian research have translated into environmental and pharmacological interventions with tremendous therapeutic potential.

Pancreatic ß cell enhancers regulate rhythmic transcription of genes controlling insulin secretion.

The mammalian transcription factors CLOCK and BMAL1 are essential components of the molecular clock that coordinate behavior and metabolism with the solar cycle. Genetic or environmental perturbation of circadian cycles contributes to metabolic disorders including type 2 diabetes. To study the impact of the cell-autonomous clock on pancreatic ß cell function, we examined pancreatic islets from mice with either intact or disrupted BMAL1 expression both throughout life and limited to adulthood. We found pronounced oscillation of insulin secretion that was synchronized with the expression of genes encoding secretory machinery and signaling factors that regulate insulin release. CLOCK/BMAL1 colocalized with the pancreatic transcription factor PDX1 within active enhancers distinct from those controlling rhythmic metabolic gene networks in liver. We also found that ß cell clock ablation in adult mice caused severe glucose intolerance. Thus, cell type-specific enhancers underlie the circadian control of peripheral metabolism throughout life and may help to explain its dysregulation in diabetes.

Circadian measurements of sirtuin biology.

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.

Circadian clock NAD+ cycle drives mitochondrial oxidative metabolism in mice.

Circadian clocks are self-sustained cellular oscillators that synchronize oxidative and reductive cycles in anticipation of the solar cycle. We found that the clock transcription feedback loop produces cycles of nicotinamide adenine dinucleotide (NAD(+)) biosynthesis, adenosine triphosphate production, and mitochondrial respiration through modulation of mitochondrial protein acetylation to synchronize oxidative metabolic pathways with the 24-hour fasting and feeding cycle. Circadian control of the activity of the NAD(+)-dependent deacetylase sirtuin 3 (SIRT3) generated rhythms in the acetylation and activity of oxidative enzymes and respiration in isolated mitochondria, and NAD(+) supplementation restored protein deacetylation and enhanced oxygen consumption in circadian mutant mice. Thus, circadian control of NAD(+) bioavailability modulates mitochondrial oxidative function and organismal metabolism across the daily cycles of fasting and feeding.

Circadian clocks and metabolism.

Circadian clocks maintain periodicity in internal cycles of behavior, physiology, and metabolism, enabling organisms to anticipate the 24-h rotation of the Earth. In mammals, circadian integration of metabolic systems optimizes energy harvesting and utilization across the light/dark cycle. Disruption of clock genes has recently been linked to sleep disorders and to the development of cardiometabolic disease. Conversely, aberrant nutrient signaling affects circadian rhythms of behavior. This chapter reviews the emerging relationship between the molecular clock and metabolic systems and examines evidence that circadian disruption exerts deleterious consequences on human health.

ROS-mediated PARP activity undermines mitochondrial function after permeability transition pore opening during myocardial ischemia-reperfusion.

BACKGROUND: Ischemia-reperfusion (I/R) studies have implicated oxidant stress, the mitochondrial permeability transition pore (mPTP), and poly(ADP-ribose) polymerase (PARP) as contributing factors in myocardial cell death. However, the interdependence of these factors in the intact, blood-perfused heart is not known. We therefore wanted to determine whether oxidant stress, mPTP opening, and PARP activity contribute to the same death pathway after myocardial I/R. METHODS AND RESULTS: A murine left anterior descending coronary artery (LAD) occlusion (30 minutes) and release (1 to 4 hours) model was employed. Experimental groups included controls and antioxidant-treated, mPTP-inhibited, or PARP-inhibited hearts. Antioxidant treatment prevented oxidative damage, mPTP opening, ATP depletion, and PARP activity, placing oxidant stress as the proximal death trigger. Genetic deletion of cyclophilin D (CypD(-/-)) prevented loss of total NAD(+) and PARP activity, and mPTP-mediated loss of mitochondrial function. Control hearts showed progressive mitochondrial depolarization and loss of ATP from 1.5 to 4 hours of reperfusion, but not outer mitochondrial membrane rupture. Neither genetic deletion of PARP-1 nor its pharmacological inhibition prevented the initial mPTP-mediated depolarization or loss of ATP, but PARP ablation did allow mitochondrial recovery by 4 hours of reperfusion. CONCLUSIONS: These results indicate that oxidant stress, the mPTP, and PARP activity contribute to a single death pathway after I/R in the heart. PARP activation undermines cell survival by preventing mitochondrial recovery after mPTP opening early in reperfusion. This suggests that PARP-mediated prolongation of mitochondrial depolarization contributes significantly to cell death via an energetic crisis rather than by mitochondrial outer membrane rupture.

Nutrient sensing and the circadian clock.

The circadian system synchronizes behavioral and physiologic processes with daily changes in the external light-dark cycle, optimizing energetic cycles with the rising and setting of the sun. Molecular clocks are organized hierarchically, with neural clocks orchestrating the daily switch between periods of feeding and fasting, and peripheral clocks generating 24h oscillations of energy storage and utilization. Recent studies indicate that clocks respond to nutrient signals and that a high-fat diet influences the period of locomotor activity under free-running conditions, a core property of the clock. A major goal is to identify the molecular basis for the reciprocal relation between metabolic and circadian pathways. Here the role of peptidergic hormones and macromolecules as nutrient signals integrating circadian and metabolic systems is highlighted.