Clara Bien Peek

Circadian Clock NAD+ Cycle Drives Mitochondrial Oxidative Metabolism in Mice

Introduction The circadian clock is a transcriptional oscillator that is thought to couple internal energetic processes with the solar cycle. Circadian oscillation in activity of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in nicotinamide adenine dinucleotide (NAD+) biosynthesis, feeds back to regulate activity of the deacetylase SIRT1 and transcription of genes encoding core clock components. Despite evidence that NAD+-dependent enzymes are important in fasting and oxidative metabolism, it is not known how the circadian cycle might affect this process. We investigated the role of clock control of NAD+ in mitochondrial dynamics and energy production. Circadian regulation of NAD+ biosynthesis synchronizes mitochondrial bioenergetics with the light-dark cycle. The core molecular clock is a transcription-translation oscillator composed of activators (CLOCK/BMAL1) that induce transcription of their own repressors (PER/CRY). Clock control of expression of the NAD+ biosynthetic enzyme NAMPT generates 24-hour variation of activity of the mitochondrial deacetylase SIRT3 and oxygen consumption. Rhythmic NAD+ oscillation couples mitochondrial bioenergetics with the light-dark cycle. Methods We determined the circadian variation in mitochondrial function by examining the adaptive response to fasting in liver of wild-type and circadian mutant mice. Quantitative analyses of NAD+ biosynthesis, lipid and glucose oxidation, and acetylation of mitochondrial proteins were performed across the circadian cycle in circadian mutant mice and in cell-based systems. Proteins displaying increased acetylation in Bmal1 mutant liver were identified by mass spectrometry, and SIRT3 activity was evaluated using label-free self-assembled monolayer and matrix desorption ionization (SAMDI) mass spectrometry in liver lysate from Bmal1 and Sirt3 knockout mice. The role of NAD+ deficiency in SIRT3 activity, mitochondrial protein acetylation, lipid oxidation, and oxygen consumption was evaluated after intraperitoneal administration of the NAD+ precursor NMN to raise NAD+ levels in Bmal1 mutant and wild-type mice. Results Lipid oxidation and mitochondrial protein acetylation exhibited circadian oscillations that corresponded with the clock-driven NAD+ cycle in mouse liver. Rhythmic NAD+ and oxidative cycles were self-sustained in fasted mice and in C2C12 myotubes, demonstrating clock control of mitochondrial function even when nutrient state remained constant. Transcription of glycolytic genes was antiphasic to lipid oxidation rhythms, and glycolytic gene expression and lactate production were increased in Bmal1–/– fibroblasts, whereas the converse occurred in Cry1–/–;Cry2–/– mutants. Lack of Bmal1 in liver led to decreased SIRT3 activity and increased mitochondrial protein acetylation, resulting in reduced function of oxidative enzymes. Finally, NAD+ supplementation with NMN restored protein deacetylation of SIRT3 targets and enhanced mitochondrial function in circadian mutant mice. Discussion Mitochondria are central to energy homeostasis in eukaryotes, and our results show that the circadian clock generates oscillations in mitochondrial oxidative capacity through rhythmic regulation of NAD+ biosynthesis. The clock thereby facilitates oxidative rhythms that correspond with the fasting-feeding cycle to maximize energy production during rest. Use of NAD+ as a central node in coupling circadian and metabolic cycles provides a rapid and reversible mechanism to augment mitochondrial oxidative function at the appropriate time in the light-dark cycle. Dinner Time! Biological clocks allow organisms to anticipate cycles of feeding, activity, and rest so that metabolic enzymes in mitochondria are ready when needed. Peek et al. (10.1126/science.1243417, published online 19 September; see the Perspective by Rey and Reddy) describe a mechanism by which the biochemical elements of the circadian clock are linked to such control of mitochondrial metabolism. The clock controls rhythmic transcription of the gene encoding the rate-limiting enzyme required for synthesis of nicotinamide adenine dinucleotide (NAD+). The concentration of NAD+ in mitochondria determines the activity of the deacetylase SIRT3, which then controls acetylation and activity of key metabolic enzymes. NAD+ also influences clock function, and thus appears to be a versatile point at which regulation of oxidative metabolism is coordinated with the daily cycles of energy consumption. The coenzyme nicotinamide adenine dinucleotide mechanistically links the circadian clock to control of energy production by mitochondria. [Also see Perspective by Rey and Reddy] 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.

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

The clockwork of insulin release In healthy people, blood glucose levels are maintained within a narrow range by several physiological mechanisms. Key among them is the release of the hormone insulin by pancreatic β cells, which occurs when glucose levels rise after a meal. In response to insulin, blood glucose is taken up by tissues that need fuel, such as muscle. β cells can anticipate the bodys varying demand for insulin throughout the 24-hour day because they have their own circadian clock. How this clock controls insulin release has been unclear. Perelis et al. now show that the activity of transcriptional enhancers specific to β cells regulates the rhythmic expression of genes involved in the assembly and trafficking of insulin secretory vesicles (see the Perspective by Dibner and Schibler). Science, this issue p. 10.1126/science.aac4250; see also p. 628 Circadian control of insulin release is mediated by transcriptional enhancers active specifically in pancreatic β cells. [Also see Perspective by Dibner and Schibler] INTRODUCTION The circadian clock is a molecular oscillator that coordinates behavior and physiology in anticipation of the daily light cycle. Desynchrony of circadian cycles, through genetic or environmental perturbation, contributes to metabolic disorders such as cardiovascular disease, obesity, and type 2 diabetes. We previously showed that disruption of the clock transcription factors CLOCK and BMAL1 in the pancreas causes hypoinsulinemic diabetes in mice. The mechanism(s) linking clock dysfunction to pancreatic β cell failure and the means by which CLOCK and BMAL1 affect glucose metabolism in the whole organism are not well understood. RATIONALE The circadian system helps to maintain glucose homeostasis across the sleep-wake cycle. This system requires cross-talk between the master clock in the central nervous system, which coordinates feeding and sleep, and peripheral tissue clocks, which synchronize behavior with the storage, mobilization, and synthesis of glucose. Although it is clear that clocks within distinct organs participate in glucose turnover, the molecular basis for time-of-day variation in organismal glucose responsiveness is still not understood. Here, we combined genome-wide analyses with gene targeting in mice to study the impact of the cell-autonomous clock on β cell function. RESULTS We found that cell-autonomous expression of CLOCK and BMAL1 in pancreatic islets isolated from wild-type mice generates robust 24-hour rhythms of glucose- and potassium chloride–stimulated insulin secretion ex vivo. About 27% of the β cell transcriptome exhibited circadian oscillation. Many of these transcripts correspond to genes coding for proteins that are involved in the assembly, trafficking, and membrane fusion of vesicles that participate in insulin secretion. Chromatin immunoprecipitation sequencing revealed that CLOCK and BMAL1 regulate cycling genes in β cells by binding at distal regulatory elements distinct from those controlling the circadian transcription of metabolic gene networks within the liver. The regulatory sites of cycling genes in the β cell resided primarily within transcriptionally active enhancers that were also bound by the pancreatic transcription factor PDX1. Finally, we found that in islets from adult mice, Bmal1 ablation either in vivo or ex vivo abrogates nutrient-responsive insulin secretion, demonstrating clock control of pancreatic β cell function throughout adult life. CONCLUSION Our results show that local clock-driven genomic rhythms program cell function across the light-dark cycle, including the priming of insulin secretion within limited time windows each day. Cell type–specific transcriptional regulation by the clock localizes to rhythmic enhancers that are unique to the β cell. Thus, our findings uncover a transcriptional process through which the core clock aligns physiology with the light cycle, revealing pathways that are important in both health and disease states such as type 2 diabetes. β cell–specific enhancers control the rhythmic transcription of genes linked to insulin secretion. Peripheral clocks maintain glucose homeostasis across the sleep-wake cycle by gating β cell insulin secretion through genome-wide transcriptional control of the assembly and trafficking of insulin secretory vesicles. Clock transcription factors bind within cell type–specific enhancer neighborhoods of cycling genes, revealing the mechanisms that synchronize rhythmic metabolism at transcriptional and physiologic levels across the light-dark cycle. 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 β 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 β 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 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.

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.

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.

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.

Circadian regulation of cellular physiology

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.

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.

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.

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.HRD1 is an E3 ligase known to play a role in targeting degradation of misfolded proteins in the ER. Here the authors show that HRD1 interacts with metabolic enzymes and its liver specific deficiency results in lower body weight, blood glucose and plasma lipids during high fat diet in mice.