mTOR regulation of metabolism limits LPS-induced monocyte inflammatory and procoagulant responses.

Translocated lipopolysaccharide (LPS) activates monocytes via TLR4 and is hypothesized to increase cardiovascular disease risk in persons living with HIV. We tested whether mTOR activity supports LPS-stimulated monocyte production of pro-inflammatory cytokines and tissue factor (TF), as it propels the inflammatory response in several immune cell types besides monocytes. However, multi-omics analyses here demonstrate that mTOR activates a metabolic pathway that limits abundance of these gene products in monocytes. Treatment of primary human monocytes with catalytic mTOR inhibitors (mTORi) increased LPS-induced polyfunctional responses, including production of IL-1ß, IL-6, and the pro-coagulant, TF. NF-κB-driven transcriptional activity is enhanced with LPS stimulation after mTORi treatment to increase expression of F3 (TF). Moreover, intracellular NAD+ availability is restricted due to decreased salvage pathway synthesis. These results document mTOR-mediated restraint of the LPS-induced transcriptional response in monocytes and a metabolic mechanism informing strategies to reverse enhanced risk of coagulopathy in pro-inflammatory states.

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.

Phosphatase of Regenerating Liver-1 Selectively Times Circadian Behavior in Darkness via Function in PDF Neurons and Dephosphorylation of TIMELESS.

The timing of behavior under natural light-dark conditions is a function of circadian clocks and photic input pathways, but a mechanistic understanding of how these pathways collaborate in animals is lacking. Here we demonstrate in Drosophila that the Phosphatase of Regenerating Liver-1 (PRL-1) sets period length and behavioral phase gated by photic signals. PRL-1 knockdown in PDF clock neurons dramatically lengthens circadian period. PRL-1 mutants exhibit allele-specific interactions with the light- and clock-regulated gene timeless (tim). Moreover, we show that PRL-1 promotes TIM accumulation and dephosphorylation. Interestingly, the PRL-1 mutant period lengthening is suppressed in constant light, and PRL-1 mutants display a delayed phase under short, but not long, photoperiod conditions. Thus, our studies reveal that PRL-1-dependent dephosphorylation of TIM is a core mechanism of the clock that sets period length and phase in darkness, enabling the behavioral adjustment to change day-night cycles.

A role for alternative splicing in circadian control of exocytosis and glucose homeostasis.

The circadian clock is encoded by a negative transcriptional feedback loop that coordinates physiology and behavior through molecular programs that remain incompletely understood. Here, we reveal rhythmic genome-wide alternative splicing (AS) of pre-mRNAs encoding regulators of peptidergic secretion within pancreatic ß cells that are perturbed in Clock-/- and Bmal1-/- ß-cell lines. We show that the RNA-binding protein THRAP3 (thyroid hormone receptor-associated protein 3) regulates circadian clock-dependent AS by binding to exons at coding sequences flanking exons that are more frequently skipped in clock mutant ß cells, including transcripts encoding Cask (calcium/calmodulin-dependent serine protein kinase) and Madd (MAP kinase-activating death domain). Depletion of THRAP3 restores expression of the long isoforms of Cask and Madd, and mimicking exon skipping in these transcripts through antisense oligonucleotide delivery in wild-type islets reduces glucose-stimulated insulin secretion. Finally, we identify shared networks of alternatively spliced exocytic genes from islets of rodent models of diet-induced obesity that significantly overlap with clock mutants. Our results establish a role for pre-mRNA alternative splicing in ß-cell function across the sleep/wake cycle.

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.

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.

Requirement for NF-κB in maintenance of molecular and behavioral circadian rhythms in mice.

The mammalian circadian clock is encoded by an autoregulatory transcription feedback loop that drives rhythmic behavior and gene expression in the brain and peripheral tissues. Transcriptomic analyses indicate cell type-specific effects of circadian cycles on rhythmic physiology, although how clock cycles respond to environmental stimuli remains incompletely understood. Here, we show that activation of the inducible transcription factor NF-κB in response to inflammatory stimuli leads to marked inhibition of clock repressors, including the Period, Cryptochrome, and Rev-erb genes, within the negative limb. Furthermore, activation of NF-κB relocalizes the clock components CLOCK/BMAL1 genome-wide to sites convergent with those bound by NF-κB, marked by acetylated H3K27, and enriched in RNA polymerase II. Abrogation of NF-κB during adulthood alters the expression of clock repressors, disrupts clock-controlled gene cycles, and impairs rhythmic activity behavior, revealing a role for NF-κB in both unstimulated and activated conditions. Together, these data highlight NF-κB-mediated transcriptional repression of the clock feedback limb as a cause of circadian disruption in response to inflammation.

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.

Usf1, a suppressor of the circadian Clock mutant, reveals the nature of the DNA-binding of the CLOCK:BMAL1 complex in mice.

Genetic and molecular approaches have been critical for elucidating the mechanism of the mammalian circadian clock. Here, we demonstrate that the ClockΔ19 mutant behavioral phenotype is significantly modified by mouse strain genetic background. We map a suppressor of the ClockΔ19 mutation to a ∼900 kb interval on mouse chromosome 1 and identify the transcription factor, Usf1, as the responsible gene. A SNP in the promoter of Usf1 causes elevation of its transcript and protein in strains that suppress the Clock mutant phenotype. USF1 competes with the CLOCK:BMAL1 complex for binding to E-box sites in target genes. Saturation binding experiments demonstrate reduced affinity of the CLOCKΔ19:BMAL1 complex for E-box sites, thereby permitting increased USF1 occupancy on a genome-wide basis. We propose that USF1 is an important modulator of molecular and behavioral circadian rhythms in mammals. DOI:http://dx.doi.org/10.7554/eLife.00426.001.

Competing E3 ubiquitin ligases govern circadian periodicity by degradation of CRY in nucleus and cytoplasm.

Period determination in the mammalian circadian clock involves the turnover rate of the repressors CRY and PER. We show that CRY ubiquitination engages two competing E3 ligase complexes that either lengthen or shorten circadian period in mice. Cloning of a short-period circadian mutant, Past-time, revealed a glycine to glutamate missense mutation in Fbxl21, an F-box protein gene that is a paralog of Fbxl3 that targets the CRY proteins for degradation. While loss of function of FBXL3 leads to period lengthening, mutation of Fbxl21 causes period shortening. FBXL21 forms an SCF E3 ligase complex that slowly degrades CRY in the cytoplasm but antagonizes the stronger E3 ligase activity of FBXL3 in the nucleus. FBXL21 plays a dual role: protecting CRY from FBXL3 degradation in the nucleus and promoting CRY degradation within the cytoplasm. Thus, the balance and cellular compartmentalization of competing E3 ligases for CRY determine circadian period of the clock in mammals.

Block of hERG K+ channel and prolongation of action potential duration by fluphenazine at submicromolar concentration.

Fluphenazine is a potent antipsychotic drug that can increase action potential duration and induce QT prolongation in several animal models and in humans. As the block of cardiac human ether-a-go-go-related gene (hERG) channels is one of the leading causes of acquired long QT syndrome, we investigated the acute effects of fluphenazine on hERG channels to determine the electrophysiological basis for its proarrhythmic potential. Fluphenazine at concentrations of 0.1-1.0 µM increased the action potential duration at 90% of repolarization (APD90) and action potential duration at 50% of repolarization (APD50) in 5 min when action potentials were elicited under current-clamp conditions in guinea pig ventricular myocytes. We examined the effects of fluphenazine on hERG channels expressed in Xenopus oocytes and HEK293 cells using two-microelectrode voltage-clamp and patch-clamp techniques. The IC50 for the fluphenazine-induced block of hERG currents in HEK293 cells at 36 °C was 0.102 µM at +20 mV. Fluphenazine-induced a concentration-dependent decrease of the current amplitude at the end of the voltage steps and hERG tail currents. The fluphenazine-dependent hERG block in Xenopus oocytes increased progressively relative to the degree of depolarization. Fluphenazine affected the channels in the activated and inactivated states but not in the closed states, and the S6 domain mutation from tyrosine to alanine at amino acid 652 (Y652A) attenuated the hERG current block. These results suggest that the antipsychotic drug fluphenazine is a potent blocker of hERG channels, providing a molecular mechanism for the drug-induced arrhythmogenic side effects.

Brain-specific rescue of Clock reveals system-driven transcriptional rhythms in peripheral tissue.

The circadian regulatory network is organized in a hierarchical fashion, with a central oscillator in the suprachiasmatic nuclei (SCN) orchestrating circadian oscillations in peripheral tissues. The nature of the relationship between central and peripheral oscillators, however, is poorly understood. We used the tetOFF expression system to specifically restore Clock function in the brains of Clock(Δ19) mice, which have compromised circadian clocks. Rescued mice showed normal locomotor rhythms in constant darkness, with activity period lengths approximating wildtype controls. We used microarray analysis to assess whether brain-specific rescue of circadian rhythmicity was sufficient to restore circadian transcriptional output in the liver. Compared to Clock mutants, Clock-rescue mice showed significantly larger numbers of cycling transcripts with appropriate phase and period lengths, including many components of the core circadian oscillator. This indicates that the SCN oscillator overcomes local circadian defects and signals directly to the molecular clock. Interestingly, the vast majority of core clock genes in liver were responsive to Clock expression in the SCN, suggesting that core clock genes in peripheral tissues are intrinsically sensitive to SCN cues. Nevertheless, most circadian output in the liver was absent or severely low-amplitude in Clock-rescue animals, demonstrating that the majority of peripheral transcriptional rhythms depend on a fully functional local circadian oscillator. We identified several new system-driven rhythmic genes in the liver, including Alas1 and Mfsd2. Finally, we show that 12-hour transcriptional rhythms (i.e., circadian "harmonics") are disrupted by Clock loss-of-function. Brain-specific rescue of Clock converted 12-hour rhythms into 24-hour rhythms, suggesting that signaling via the central circadian oscillator is required to generate one of the two daily peaks of expression. Based on these data, we conclude that 12-hour rhythms are driven by interactions between central and peripheral circadian oscillators.

Second-generation high-throughput forward genetic screen in mice to isolate subtle behavioral mutants.

Forward genetic screens have been highly successful in revealing roles of genes and pathways in complex biological events. Traditionally these screens have focused on isolating mutants with the greatest phenotypic deviance, with the hopes of discovering genes that are central to the biological event being investigated. Behavioral screens in mice typically use simple activity-based assays as endophenotypes for more complex emotional states of the animal. They generally set the selection threshold for a putative mutant at 3 SDs (z score of 3) from the average behavior of normal animals to minimize false-positive results. Behavioral screens using a high threshold for detection have generally had limited success, with high false-positive rates and subtle phenotypic differences that have made mapping and cloning difficult. In addition, targeted reverse genetic approaches have shown that when genes central to behaviors such as open field behavior, psychostimulant response, and learning and memory tasks are mutated, they produce subtle phenotypes that differ from wild-type animals by 1 to 2 SDs (z scores of 1 to 2). We have conducted a second-generation (G2) dominant N-ethyl-N-nitrosourea (ENU) screen especially designed to detect subtle behavioral mutants for open field activity and psychostimulant response behaviors. We successfully detect mutant lines with only 1 to 2 SD shifts in mean response compared with wild-type control animals and present a robust statistical and methodological framework for conducting such forward genetic screens. Using this methodology we have screened 229 ENU mutant lines and have identified 15 heritable mutant lines. We conclude that for screens in mice that use activity-based endophenotypic measurements for complex behavioral states, this G2 screening approach yields better results.

Generation of N-ethyl-N-nitrosourea (ENU) diabetes models in mice demonstrates genotype-specific action of glucokinase activators.

We performed genome-wide mutagenesis in C57BL/6J mice using N-ethyl-N-nitrosourea to identify mutations causing high blood glucose early in life and to produce new animal models of diabetes. Of a total of 13 new lines confirmed by heritability testing, we identified two semi-dominant pedigrees with novel missense mutations (Gck(K140E) and Gck(P417R)) in the gene encoding glucokinase (Gck), the mammalian glucose sensor that is mutated in human maturity onset diabetes of the young type 2 and the target of emerging anti-hyperglycemic agents that function as glucokinase activators (GKAs). Diabetes phenotype corresponded with genotype (mild-to-severe: Gck(+/+) < Gck(P417R/+), Gck(K140E)(/+) < Gck(P417R/P417R), Gck(P417R/K140E), and Gck(K140E/K140E)) and with the level of expression of GCK in liver. Each mutant was produced as the recombinant enzyme in Escherichia coli, and analysis of k(cat) and tryptophan fluorescence (I(320/360)) during thermal shift unfolding revealed a correlation between thermostability and the severity of hyperglycemia in the whole animal. Disruption of the glucokinase regulatory protein-binding site (GCK(K140E)), but not the ATP binding cassette (GCK(P417R)), prevented inhibition of enzyme activity by glucokinase regulatory protein and corresponded with reduced responsiveness to the GKA drug. Surprisingly, extracts from liver of diabetic GCK mutants inhibited activity of the recombinant enzyme, a property that was also observed in liver extracts from mice with streptozotocin-induced diabetes. These results indicate a relationship between genotype, phenotype, and GKA efficacy. The integration of forward genetic screening and biochemical profiling opens a pathway for preclinical development of mechanism-based diabetes therapies.

Block of the human ether-a-go-go-related gene (hERG) K+ channel by the antidepressant desipramine.

Desipramine is a tricyclic antidepressant for psychiatric disorders that can induce QT prolongation, which may lead to torsades de pointes. Since blockade of cardiac human ether-a-go-go-related gene (hERG) channels is an important cause of acquired long QT syndrome, we investigated the acute effects of desipramine on hERG channels to determine the electrophysiological basis for its pro-arrhythmic potential. We examined the effects of desipramine on the hERG channels expressed in Xenopus oocytes using two-microelectrode voltage-clamp techniques. Desipramine-induced concentration-dependent decreases in the current amplitude at the end of the voltage steps and hERG tail currents. The IC(50) for desipramine needed to block the hERG current in Xenopus oocytes decreased progressively relative to the degree of depolarization. Desipramine affected the channels in the activated and inactivated states but not in the closed states. The S6 domain mutations, Tyr-652 located in the S6 domain of the hERG channel reduced the potency of the channel block by desipramine more than a mutation of Phe-656 in the same region. These results suggest that desipramine is a blocker of the hERG channels, providing a molecular mechanism for the arrhythmogenic side effects during the clinical administration of desipramine.

Inhibition of the human ether-a-go-go-related gene (HERG) K+ channels by Lindera erythrocarpa.

Lindera erythrocarpa Makino (Lauraceae) is used as a traditional medicine for analgesic, antidote, and antibacterial purposes and shows anti-tumor activity. We studied the effects of Lindera erythrocarpa on the human ether-a-go-go-related gene (HERG) channel, which appears of importance in favoring cancer progression in vivo and determining cardiac action potential duration. Application of MeOH extract of Lindera erythrocarpa showed a dose-dependent decrease in the amplitudes of the outward currents measured at the end of the pulse (I(HERG)) and the tail currents of HERG (I(tail)). When the BuOH fraction and H(2)O fraction of Lindera erythrocarpa were added to the perfusate, both I(HERG) and I(tail) were suppressed, while the hexane fraction, CHCl(3) fraction, and EtOAc fraction did not inhibit either I(HERG) or I(tail). The potential required for half-maximal activation caused by EtOAc fraction, BuOH fraction, and H(2)O fraction shifted significantly. The BuOH fraction and H(2)O fraction (100 microg/mL) decreased g(max) by 59.6% and 52.9%, respectively. The H(2)O fraction- and BuOH fraction-induced blockades of I(tail) progressively decreased with increasing depolarization, showing the voltage-dependent block. Our findings suggest that Lindera erythrocarpa, a traditional medicine, blocks HERG channel, which could contribute to its anticancer and cardiac arrhythmogenic effect.

Block of HERG k channel by classic histamine h(1) receptor antagonist chlorpheniramine.

Chlorpheniramine is a potent first-generation histamine H(1) receptor antagonist that can increase action potential duration and induce QT prolongation in several animal models. Since block of cardiac human ether-a-go-go-related gene (hERG) channels is one of leading causes of acquired long QT syndrome, we investigated the acute effects of chlorpheniramine on hERG channels to determine the electrophysiological basis for its proarrhythmic potential. We examined the effects of chlorpheniramine on the hERG channels expressed in Xenopus oocytes using two-microelectrode voltage-clamp techniques. Chlorpheniramine induced a concentration-dependent decrease of the current amplitude at the end of the voltage steps and hERG tail currents. The IC(50) of chlorpheniramine-dependent hERG block in Xenopus oocytes decreased progressively relative to the degree of depolarization. Chlorpheniramine affected the channels in the activated and inactivated states but not in the closed states. The S6 domain mutations Y652A and F656A partially attenuated (Y652A) or abolished (F656A) the hERG current block. These results suggest that the H(1) antihistamine, chlorpheniramine is a blocker of the hERG channels, providing a molecular mechanism for the drug-induced arrhythmogenic side effects.

H(1) antihistamine drug promethazine directly blocks hERG K(+) channel.

Promethazine is a phenothiazine derivative with antihistaminic (H(1)), sedative, antiemetic, anticholinergic, and antimotion sickness properties that can induce QT prolongation, which may lead to torsades de pointes. Since block of cardiac human ether-a-go-go-related gene (hERG) channels is one of the leading causes of acquired long QT syndrome, we investigated the acute effects of promethazine on hERG channels to determine the electrophysiological basis for its proarrhythmic potential. Promethazine increased the action potential duration at 90% of repolarization (APD(90)) in a concentration-dependent manner, with an IC(50) of 0.73microM when action potentials were elicited under current clamp in guinea pig ventricular myocytes. We examined the effects of promethazine on the hERG channels expressed in Xenopus oocytes and HEK293 cells using two-microelectrode voltage-clamp and patch-clamp techniques. Promethazine induced a concentration-dependent decrease of the current amplitude at the end of the voltage steps and hERG tail currents. The IC(50) of promethazine dependent hERG block in Xenopus oocytes decreased progressively relative to the degree of depolarization. The IC(50) for the promethazine-induced block of the hERG currents in HEK293 cells at 36 degrees C was 1.46microM at +20mV. Promethazine affected the channels in the activated and inactivated states but not in the closed states. The S6 domain mutations, Y652A and F656A partially attenuated (Y652A) or abolished (F656A) the hERG current block. These results suggest that promethazine is a blocker of the hERG channels, providing a molecular mechanism for the arrhythmogenic side effects during the clinical administration of promethazine.