Misaligned feeding uncouples daily rhythms within brown adipose tissue and between peripheral clocks.

Extended food consumption during the rest period perturbs the phase relationship between circadian clocks in the periphery and the brain, leading to adverse health effects. Beyond the liver, how metabolic organs respond to a timed hypocaloric diet is largely unexplored. We investigated how feeding schedules impacted circadian gene expression in epididymal white and brown adipose tissue (eWAT and BAT) compared to the liver and hypothalamus. We restricted food to either daytime or nighttime in C57BL/6J male mice, with or without caloric restriction. Unlike the liver and eWAT, rhythmic clock genes in the BAT remained insensitive to feeding time, similar to the hypothalamus. We uncovered an internal split within the BAT in response to conflicting environmental cues, displaying inverted oscillations on a subset of metabolic genes without modifying its local core circadian machinery. Integrating tissue-specific responses on circadian transcriptional networks with metabolic outcomes may help elucidate the mechanism underlying the health burden of eating at unusual times.

Metabolic and chemical architecture of the mammalian circadian clock.

Circadian rhythms are endogenous periodic biological processes that occur on a daily timescale. These rhythms are generated by a transcriptional/translational feedback loop that consists of the CLOCK-BMAL1 heterodimeric transcriptional activator complex and the PER1/2-CRY1/2-CK1δ/ε repressive complex. The output pathways of this molecular feedback loop generate circadian rhythmicity in various biological processes. Among these, metabolism is a primary regulatory target of the circadian clock which can also feedback to modulate clock function. This intertwined relationship between circadian rhythms and metabolism makes circadian clock components promising therapeutic targets. Despite this, pharmacological therapeutics that target the circadian clock are relatively rare. In this review, we hope to stimulate interest in chemical chronobiology by providing a comprehensive background on the molecular mechanism of mammalian circadian rhythms and their connection to metabolism, highlighting important studies in the chemical approach to circadian research, and offering our perspectives on future developments in the field.

The Disordered Amino Terminus of the Circadian Enzyme Nocturnin Modulates Its NADP(H) Phosphatase Activity by Changing Protein Dynamics.

Endogenous circadian clocks control the rhythmicity of a broad range of behavioral and physiological processes, and this is entrained by the daily fluctuations in light and dark. Nocturnin (Noct) is a rhythmically expressed gene regulated by the circadian clock that belongs to the CCR4 family of endonuclease-exonuclease-phosphatase (EEP) enzymes, and the NOCT protein exhibits phosphatase activity, catalyzing the removal of the 2'-phosphate from NADP(H). In addition to its daily nighttime peak of expression, it is also induced by acute stimuli. Loss of Nocturnin (Noct-/-) in mice results in resistance to high-fat diet-induced obesity, and loss of Noct in HEK293T cells confers a protective effect to oxidative stress. Modeling of the full-length Nocturnin protein reveals a partially structured amino terminus that is disparate from its CCR4 family members. The high sequence conservation of a leucine zipper-like (LZ-like) motif, the only structural element in the amino terminus, highlights the potential importance of this domain in modulating phosphatase activity. In vitro biochemical and biophysical techniques demonstrate that the LZ-like domain within the flexible N-terminus is necessary for preserving the active site cleft in an optimal conformation to promote the efficient turnover of the substrate. This modulation occurs in cis and is pivotal in maintaining the stability and conformational integrity of the enzyme. These new findings suggest an additional layer of modulating the activity of Nocturnin in addition to its rhythmicity to provide fine-tuned control over cellular levels of NADPH.

Circadian alignment of early onset caloric restriction promotes longevity in male C57BL/6J mice.

Caloric restriction (CR) prolongs life span, yet the mechanisms by which it does so remain poorly understood. Under CR, mice self-impose chronic cycles of 2-hour feeding and 22-hour fasting, raising the question of if it is calories, fasting, or time of day that is the cause of this increased life span. We show here that 30% CR was sufficient to extend the life span by 10%; however, a daily fasting interval and circadian alignment of feeding acted together to extend life span by 35% in male C57BL/6J mice. These effects were independent of body weight. Aging induced widespread increases in gene expression associated with inflammation and decreases in the expression of genes encoding components of metabolic pathways in liver from ad libitum-fed mice. CR at night ameliorated these aging-related changes. Our results show that circadian interventions promote longevity and provide a perspective to further explore mechanisms of aging.

Time to target the circadian clock for drug discovery.

The circadian clock is an intracellular timekeeping device that drives daily rhythms in diverse and extensive processes throughout the body. The clock mechanism comprises a core transcription/translation negative feedback loop that is modulated by a complex set of additional interlocking feedback loops. Pharmacological manipulation of the clock may be valuable for treating many maladies including jet lag, shift work and related sleep disorders, various metabolic diseases, and cancer. We review recent identification of small-molecule clock modulators and discuss the biochemical features of the core clock that may be amenable to future drug discovery.

Natural antisense transcript of Period2, Per2AS, regulates the amplitude of the mouse circadian clock.

In mammals, a set of core clock genes form transcription-translation feedback loops to generate circadian oscillations. We and others recently identified a novel transcript at the Period2 (Per2) locus that is transcribed from the antisense strand of Per2 This transcript, Per2AS, is expressed rhythmically and antiphasic to Per2 mRNA, leading to our hypothesis that Per2AS and Per2 mutually inhibit each other's expression and form a double negative feedback loop. By perturbing the expression of Per2AS, we found that Per2AS transcription, but not transcript, represses Per2 However, Per2 does not repress Per2AS, as Per2 knockdown led to a decrease in the Per2AS level, indicating that Per2AS forms a single negative feedback loop with Per2 and maintains the level of Per2 within the oscillatory range. Per2AS also regulates the amplitude of the circadian clock, and this function cannot be solely explained through its interaction with Per2, as Per2 knockdown does not recapitulate the phenotypes of Per2AS perturbation. Overall, our data indicate that Per2AS is an important regulatory molecule in the mammalian circadian clock machinery. Our work also supports the idea that antisense transcripts of core clock genes constitute a common feature of circadian clocks, as they are found in other organisms.

Importance of circadian timing for aging and longevity.

Dietary restriction (DR) decreases body weight, improves health, and extends lifespan. DR can be achieved by controlling how much and/or when food is provided, as well as by adjusting nutritional composition. Because these factors are often combined during DR, it is unclear which are necessary for beneficial effects. Several drugs have been utilized that target nutrient-sensing gene pathways, many of which change expression throughout the day, suggesting that the timing of drug administration is critical. Here, we discuss how dietary and pharmacological interventions promote a healthy lifespan by influencing energy intake and circadian rhythms.

Measuring problem prescription opioid use among patients receiving long-term opioid analgesic treatment: development and evaluation of an algorithm for use in EHR and claims data.

Objective: Opioid surveillance in response to the opioid epidemic will benefit from scalable, automated algorithms for identifying patients with clinically documented signs of problem prescription opioid use. Existing algorithms lack accuracy. We sought to develop a high-sensitivity, high-specificity classification algorithm based on widely available structured health data to identify patients receiving chronic extended-release/long-acting (ER/LA) therapy with evidence of problem use to support subsequent epidemiologic investigations. Methods: Outpatient medical records of a probability sample of 2,000 Kaiser Permanente Washington patients receiving ≥60 days' supply of ER/LA opioids in a 90-day period from 1 January 2006 to 30 June 2015 were manually reviewed to determine the presence of clinically documented signs of problem use and used as a reference standard for algorithm development. Using 1,400 patients as training data, we constructed candidate predictors from demographic, enrollment, encounter, diagnosis, procedure, and medication data extracted from medical claims records or the equivalent from electronic health record (EHR) systems, and we used adaptive least absolute shrinkage and selection operator (LASSO) regression to develop a model. We evaluated this model in a comparable 600-patient validation set. We compared this model to ICD-9 diagnostic codes for opioid abuse, dependence, and poisoning. This study was registered with ClinicalTrials.gov as study NCT02667262 on 28 January 2016. Results: We operationalized 1,126 potential predictors characterizing patient demographics, procedures, diagnoses, timing, dose, and location of medication dispensing. The final model incorporating 53 predictors had a sensitivity of 0.582 at positive predictive value (PPV) of 0.572. ICD-9 codes for opioid abuse, dependence, and poisoning had a sensitivity of 0.390 at PPV of 0.599 in the same cohort. Conclusions: Scalable methods using widely available structured EHR/claims data to accurately identify problem opioid use among patients receiving long-term ER/LA therapy were unsuccessful. This approach may be useful for identifying patients needing clinical evaluation.

Spatiotemporal regulation of NADP(H) phosphatase Nocturnin and its role in oxidative stress response.

An intimate link exists between circadian clocks and metabolism with nearly every metabolic pathway in the mammalian liver under circadian control. Circadian regulation of metabolism is largely driven by rhythmic transcriptional activation of clock-controlled genes. Among these output genes, Nocturnin (Noct) has one of the highest amplitude rhythms at the mRNA level. The Noct gene encodes a protein (NOC) that is highly conserved with the endonuclease/exonuclease/phosphatase (EEP) domain-containing CCR4 family of deadenylases, but highly purified NOC possesses little or no ribonuclease activity. Here, we show that NOC utilizes the dinucleotide NADP(H) as a substrate, removing the 2' phosphate to generate NAD(H), and is a direct regulator of oxidative stress response through its NADPH 2' phosphatase activity. Furthermore, we describe two isoforms of NOC in the mouse liver. The cytoplasmic form of NOC is constitutively expressed and associates externally with membranes of other organelles, including the endoplasmic reticulum, via N-terminal glycine myristoylation. In contrast, the mitochondrial form of NOC possesses high-amplitude circadian rhythmicity with peak expression level during the early dark phase. These findings suggest that NOC regulates local intracellular concentrations of NADP(H) in a manner that changes over the course of the day.

Periodicity, repression, and the molecular architecture of the mammalian circadian clock.

Large molecular machines regulate daily cycles of transcriptional activity and help generate rhythmic behavior. In recent years, structural and biochemical analyses have elucidated a number of principles guiding the interactions of proteins that form the basis of circadian timing. In its simplest form, the circadian clock is composed of a transcription/translation feedback loop. However, this description elides a complicated process of activator recruitment, chromatin decompaction, recruitment of coactivators, expression of repressors, formation of a repressive complex, repression of the activators, and ultimately degradation of the repressors and reinitiation of the cycle. Understanding the core principles underlying the clock requires careful examination of molecular and even atomic level details of these processes. Here, we review major structural and biochemical findings in circadian biology and make the argument that shared protein interfaces within the clockwork are critical for both the generation of rhythmicity and timing of the clock.

Chemical and structural analysis of a photoactive vertebrate cryptochrome from pigeon.

Computational and biochemical studies implicate the blue-light sensor cryptochrome (CRY) as an endogenous light-dependent magnetosensor enabling migratory birds to navigate using the Earth's magnetic field. Validation of such a mechanism has been hampered by the absence of structures of vertebrate CRYs that have functional photochemistry. Here we present crystal structures of Columba livia (pigeon) CRY4 that reveal evolutionarily conserved modifications to a sequence of Trp residues (Trp-triad) required for CRY photoreduction. In ClCRY4, the Trp-triad chain is extended to include a fourth Trp (W369) and a Tyr (Y319) residue at the protein surface that imparts an unusually high quantum yield of photoreduction. These results are consistent with observations of night migratory behavior in animals at low light levels and could have implications for photochemical pathways allowing magnetosensing.

Nobiletin fortifies mitochondrial respiration in skeletal muscle to promote healthy aging against metabolic challenge.

Circadian disruption aggravates age-related decline and mortality. However, it remains unclear whether circadian enhancement can retard aging in mammals. We previously reported that the small molecule Nobiletin (NOB) activates ROR (retinoid acid receptor-related orphan receptor) nuclear receptors to potentiate circadian oscillation and protect against metabolic dysfunctions. Here we show that NOB significantly improves metabolic fitness in naturally aged mice fed with a regular diet (RD). Furthermore, NOB enhances healthy aging in mice fed with a high-fat diet (HF). In HF skeletal muscle, the NOB-ROR axis broadly activates genes for mitochondrial respiratory chain complexes (MRCs) and fortifies MRC activity and architecture, including Complex II activation and supercomplex formation. These mechanisms coordinately lead to a dichotomous mitochondrial optimization, namely increased ATP production and reduced ROS levels. Together, our study illustrates a focal mechanism by a clock-targeting pharmacological agent to optimize skeletal muscle mitochondrial respiration and promote healthy aging in metabolically stressed mammals.

Medicine in the Fourth Dimension.

The importance of circadian biology has rarely been considered in pre-clinical studies, and even more when translating to the bedside. Circadian biology is becoming a critical factor for improving drug efficacy and diminishing drug toxicity. Indeed, there is emerging evidence showing that some drugs are more effective at nighttime than daytime, whereas for others it is the opposite. This suggests that the biology of the target cell will determine how an organ will respond to a drug at a specific time of the day, thus modulating pharmacodynamics. Thus, it is now time that circadian factors become an integral part of translational research.

The Circadian Protein Nocturnin Regulates Metabolic Adaptation in Brown Adipose Tissue.

Fine-tuning of transcriptional responses can be critical for long-term outcomes in response to an environmental challenge. The circadian protein Nocturnin belongs to a family of proteins that include exonucleases, endonucleases, and phosphatases and is most closely related to the CCR4 family of deadenylases that regulate the cellular transcriptome via control of poly(A) tail length of RNA transcripts. In this study, we investigate the role of Nocturnin in regulating the transcriptional response and downstream metabolic adaptations during cold exposure in brown adipose tissue. We find that Nocturnin exhibits dual localization within the cytosol and mitochondria, and loss of Nocturnin causes changes in expression of networks of mRNAs involved in mitochondrial function. Furthermore, Nocturnin-/- animals display significantly elevated levels of tricarboxylic acid cycle intermediates, indicating that they have distinct metabolic adaptations during a prolonged cold exposure. We conclude that cold-induced stimulation of Nocturnin levels can regulate long-term metabolic adaptations to environmental challenges.

Using natural language processing of clinical text to enhance identification of opioid-related overdoses in electronic health records data.

PURPOSE: To enhance automated methods for accurately identifying opioid-related overdoses and classifying types of overdose using electronic health record (EHR) databases. METHODS: We developed a natural language processing (NLP) software application to code clinical text documentation of overdose, including identification of intention for self-harm, substances involved, substance abuse, and error in medication usage. Using datasets balanced with cases of suspected overdose and records of individuals at elevated risk for overdose, we developed and validated the application using Kaiser Permanente Northwest data, then tested portability of the application using Kaiser Permanente Washington data. Datasets were chart-reviewed to provide a gold standard for comparison and evaluation of the automated method. RESULTS: The method performed well in identifying overdose (sensitivity = 0.80, specificity = 0.93), intentional overdose (sensitivity = 0.81, specificity = 0.98), and involvement of opioids (excluding heroin, sensitivity = 0.72, specificity = 0.96) and heroin (sensitivity = 0.84, specificity = 1.0). The method performed poorly at identifying adverse drug reactions and overdose due to patient error and fairly at identifying substance abuse in opioid-related unintentional overdose (sensitivity = 0.67, specificity = 0.96). Evaluation using validation datasets yielded significant reductions, in specificity and negative predictive values only, for many classifications mentioned above. However, these measures remained above 0.80, thus, performance observed during development was largely maintained during validation. Similar results were obtained when evaluating portability, although there was a significant reduction in sensitivity for unintentional overdose that was attributed to missing text clinical notes in the database. CONCLUSIONS: Methods that process text clinical notes show promise for improving accuracy and fidelity at identifying and classifying overdoses according to type using EHR data.

Development of an algorithm to identify inpatient opioid-related overdoses and oversedation using electronic data.

PURPOSE: To facilitate surveillance and evaluate interventions addressing opioid-related overdoses, algorithms are needed for use in large health care databases to identify and differentiate community-occurring opioid-related overdoses from inpatient-occurring opioid-related overdose/oversedation. METHODS: Data were from Kaiser Permanente Northwest (KPNW), a large integrated health plan. We iteratively developed and evaluated an algorithm for electronically identifying inpatient overdose/oversedation in KPNW hospitals from 1 January 2008 to 31 December 2014. Chart audits assessed accuracy; data sources included administrative and clinical records. RESULTS: The best-performing algorithm used these rules: (1) Include events with opioids administered in an inpatient setting (including emergency department/urgent care) followed by naloxone administration within 275 hours of continuous inpatient stay; (2) exclude events with electroconvulsive therapy procedure codes; and (3) exclude events in which an opioid was administered prior to hospital discharge and followed by readmission with subsequent naloxone administration. Using this algorithm, we identified 870 suspect inpatient overdose/oversedation events and chart audited a random sample of 235. Of the random sample, 185 (78.7%) were deemed overdoses/oversedation, 37 (15.5%) were not, and 13 (5.5%) were possible cases. The number of hours between time of opioid and naloxone administration did not affect algorithm accuracy. When "possible" overdoses/oversedations were included with confirmed events, overall positive predictive value (PPV) was very good (PPV = 84.0%). Additionally, PPV was reasonable when evaluated specifically for hospital stays with emergency/urgent care admissions (PPV = 77.0%) and excellent for elective surgery admissions (PPV = 97.0%). CONCLUSIONS: Algorithm performance was reasonable for identifying inpatient overdose/oversedation with best performance among elective surgery patients.

A novel mouse model overexpressing Nocturnin results in decreased fat mass in male mice.

Nocturnin (NOCT) belongs to the Mg2+ dependent Exonucleases, Endonucleases, Phosphatase (EEP) family of enzymes that exhibit various functions in vitro and in vivo. NOCT is known to function as a deadenylase, cleaving poly-A tails from mRNA (messenger RNA) transcripts. Previously, we reported a role for NOCT in regulating bone marrow stromal cell differentiation through its interactions with PPARγ. In this study, we characterized the skeletal and adipose tissue phenotype when we globally overexpressed Noct in vivo. After 12 weeks of Noct overexpression, transgenic male mice had lower fat mass compared to controls, with no significant differences in the skeleton. Based on the presence of a mitochondrial target sequence in NOCT, we determined that mouse NOCT protein localizes to the mitochondria; subsequently, we found that NOCT overexpression led to a significant increase in the preadipocytes ability to utilize oxidative phosphorylation for ATP (adenosine triphosphate) generation. In summary, the effects of NOCT on adipocytes are likely through its novel role as a mediator of mitochondrial function.

Identifying and classifying opioid-related overdoses: A validation study.

PURPOSE: The study aims to develop and validate algorithms to identify and classify opioid overdoses using claims and other coded data, and clinical text extracted from electronic health records using natural language processing (NLP). METHODS: Primary data were derived from Kaiser Permanente Northwest (2008-2014), an integrated health care system (~n > 475 000 unique individuals per year). Data included International Classification of Diseases, Ninth Revision (ICD-9) codes for nonfatal diagnoses, International Classification of Diseases, Tenth Revision (ICD-10) codes for fatal events, clinical notes, and prescription medication records. We assessed sensitivity, specificity, positive predictive value, and negative predictive value for algorithms relative to medical chart review and conducted assessments of algorithm portability in Kaiser Permanente Washington, Tennessee State Medicaid, and Optum. RESULTS: Code-based algorithm performance was excellent for opioid-related overdoses (sensitivity = 97.2%, specificity = 84.6%) and classification of heroin-involved overdoses (sensitivity = 91.8%, specificity = 99.0%). Performance was acceptable for code-based suicide/suicide attempt classifications (sensitivity = 70.7%, specificity = 90.5%); sensitivity improved with NLP (sensitivity = 78.7%, specificity = 91.0%). Performance was acceptable for the code-based substance abuse-involved classification (sensitivity = 75.3%, specificity = 79.5%); sensitivity improved with the NLP-enhanced algorithm (sensitivity = 80.5%, specificity = 76.3%). The opioid-related overdose algorithm performed well across portability assessment sites, with sensitivity greater than 96% and specificity greater than 84%. Cross-site sensitivity for heroin-involved overdose was greater than 87%, specificity greater than or equal to 99%. CONCLUSIONS: Code-based algorithms developed to detect opioid-related overdoses and classify them according to heroin involvement perform well. Algorithms for classifying suicides/attempts and abuse-related opioid overdoses perform adequately for use for research, particularly given the complexity of classifying such overdoses. The NLP-enhanced algorithms for suicides/suicide attempts and abuse-related overdoses perform significantly better than code-based algorithms and are appropriate for use in settings that have data and capacity to use NLP.