Biliana Marcheva

Disruption of the clock components CLOCK and BMAL1 leads to hypoinsulinaemia and diabetes

The molecular clock maintains energy constancy by producing circadian oscillations of rate-limiting enzymes involved in tissue metabolism across the day and night. During periods of feeding, pancreatic islets secrete insulin to maintain glucose homeostasis, and although rhythmic control of insulin release is recognized to be dysregulated in humans with diabetes, it is not known how the circadian clock may affect this process. Here we show that pancreatic islets possess self-sustained circadian gene and protein oscillations of the transcription factors CLOCK and BMAL1. The phase of oscillation of islet genes involved in growth, glucose metabolism and insulin signalling is delayed in circadian mutant mice, and both Clock and Bmal1 (also called Arntl) mutants show impaired glucose tolerance, reduced insulin secretion and defects in size and proliferation of pancreatic islets that worsen with age. Clock disruption leads to transcriptome-wide alterations in the expression of islet genes involved in growth, survival and synaptic vesicle assembly. Notably, conditional ablation of the pancreatic clock causes diabetes mellitus due to defective β-cell function at the very latest stage of stimulus–secretion coupling. These results demonstrate a role for the β-cell clock in coordinating insulin secretion with the sleep–wake cycle, and reveal that ablation of the pancreatic clock can trigger the onset of diabetes mellitus.

Circadian clock feedback cycle through NAMPT-mediated NAD+ biosynthesis.

Circadian Oscillations The 24-hour day-night cycle plays an important role in mammalian physiology and behavior and, as most travelers are well aware, there is an intimate link between our in-built circadian clocks and metabolic rhythms. This link is in part forged by the protein deacetylase SIRT1, which regulates the clocks molecular circuitry. SIRT1 uses as a cofactor the cellular metabolite NAD+, which is synthesized through a salvage pathway that includes the enzyme nicotinamide phosphoribosyltransferase (NAMPT) (see the Perspective by Wijnen). Ramsey et al. (p. 651; published online 19 March) and Nakahata et al. (p. 654, published online 12 March) now show that NAMPT and NAD+ levels oscillate during the daily 24-hour cycle and that this oscillation is regulated by the circadian clock. Furthermore, the oscillations in NAD+ modulate the activity of SIRT1 feeding back into the circadian clock. A transcriptional-enzymatic feedback loop controls interactions between metabolism and circadian rhythms in mouse cells. The circadian clock is encoded by a transcription-translation feedback loop that synchronizes behavior and metabolism with the light-dark cycle. Here we report that both the rate-limiting enzyme in mammalian nicotinamide adenine dinucleotide (NAD+) biosynthesis, nicotinamide phosphoribosyltransferase (NAMPT), and levels of NAD+ display circadian oscillations that are regulated by the core clock machinery in mice. Inhibition of NAMPT promotes oscillation of the clock gene Per2 by releasing CLOCK:BMAL1 from suppression by SIRT1. In turn, the circadian transcription factor CLOCK binds to and up-regulates Nampt, thus completing a feedback loop involving NAMPT/NAD+ and SIRT1/CLOCK:BMAL1.

Circadian rhythms, sleep, and metabolism

The discovery of the genetic basis for circadian rhythms has expanded our knowledge of the temporal organization of behavior and physiology. The observations that the circadian gene network is present in most living organisms from eubacteria to humans, that most cells and tissues express autonomous clocks, and that disruption of clock genes results in metabolic dysregulation have revealed interactions between metabolism and circadian rhythms at neural, molecular, and cellular levels. A major challenge remains in understanding the interplay between brain and peripheral clocks and in determining how these interactions promote energy homeostasis across the sleep-wake cycle. In this Review, we evaluate how investigation of molecular timing may create new opportunities to understand and develop therapies for obesity and diabetes.

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.

Clock genes and metabolic disease

The circadian system is a key integrator of behavior and metabolism that synchronizes physiological processes with the rotation of the Earth on its axis. In mammals, the clock is present not only within the central pacemaker neurons of the hypothalamus, but also within extra-suprachiasmatic nucleus (SCN) regions of brain and nearly all peripheral tissues. Recent evidence suggests that the complex feedback networks that encompass both the circadian and metabolic systems are intimately intertwined and that disruption of either system leads to reciprocal disturbances in the other. We anticipate that improved understanding of the interconnections between the circadian and metabolic networks will open new windows on the treatment of sleep and metabolic disorders, including diabetes mellitus and obesity.

Gamma-protocadherins regulate the functional integrity of hypothalamic feeding circuitry in mice.

The hypothalamic neuronal circuits that modulate energy homeostasis become mature and functional during early postnatal life. However, the molecular mechanism underlying this developmental process remains largely unknown. Here we use a mouse genetic approach to investigate the role of gamma-protocadherins (Pcdh-gammas) in hypothalamic neuronal circuits. First, we show that rat insulin promoter (RIP)-Cre conditional knockout mice lacking Pcdh-gammas in a broad subset of hypothalamic neurons are obese and hyperphagic. Second, specific deletion of Pcdh-gammas in anorexigenic proopiomelanocortin (POMC) expressing neurons also leads to obesity. Using cell lineage tracing, we show that POMC and RIP-Cre expressing neurons do not overlap but interact with each other in the hypothalamus. Moreover, excitatory synaptic inputs are reduced in Pcdh-gamma deficient POMC neurons. Genetic evidence from both knockout models shows that Pcdh-gammas can regulate POMC neuronal function autonomously and non-autonomously through cell-cell interaction. Taken together, our data demonstrate that Pcdh-gammas regulate the formation and functional integrity of hypothalamic feeding circuitry in mice.

Circadian genes and insulin exocytosis

The molecular clock controls 24-hour cycles of behavioral and physiological processes across the day-night cycle. Disruption of circadian rhythmicity has been implicated in the pathogenesis of several diseases, including the metabolic syndrome, although the role of clock genes in these disorders is still not well understood. Studies of the etiology of diabetes in circadian mutant mice have revealed a novel role for the clock in pancreatic β-cell insulin secretion, suggesting that a major cellular function of the circadian network involves control of protein exocytosis.