Sexual Dimorphism in Adipose-Hypothalamic Crosstalk and the Contribution of Aryl Hydrocarbon Receptor to Regulate Energy Homeostasis.

There are fundamental sex differences in the regulation of energy homeostasis. Better understanding of the underlying mechanisms of energy balance that account for this asymmetry will assist in developing sex-specific therapies for sexually dimorphic diseases such as obesity. Multiple organs, including the hypothalamus and adipose tissue, play vital roles in the regulation of energy homeostasis, which are regulated differently in males and females. Various neuronal populations, particularly within the hypothalamus, such as arcuate nucleus (ARC), can sense nutrient content of the body by the help of peripheral hormones such leptin, derived from adipocytes, to regulate energy homeostasis. This review summarizes how adipose tissue crosstalk with homeostatic network control systems in the brain, which includes energy regulatory regions and the hypothalamic-pituitary axis, contribute to energy regulation in a sex-specific manner. Moreover, development of obesity is contingent upon diet and environmental factors. Substances from diet and environmental contaminants can exert insidious effects on energy metabolism, acting peripherally through the aryl hydrocarbon receptor (AhR). Developmental AhR activation can impart permanent alterations of neuronal development that can manifest a number of sex-specific physiological changes, which sometimes become evident only in adulthood. AhR is currently being investigated as a potential target for treating obesity. The consensus is that impaired function of the receptor protects from obesity in mice. AhR also modulates sex steroid receptors, and hence, one of the objectives of this review is to explain why investigating sex differences while examining this receptor is crucial. Overall, this review summarizes sex differences in the regulation of energy homeostasis imparted by the adipose-hypothalamic axis and examines how this axis can be affected by xenobiotics that signal through AhR.

Mechanisms of circadian clock interactions with aryl hydrocarbon receptor signalling.

Per-Arnt-Sim (PAS) domain-containing proteins are critical to homeostatic regulatory networks that mediate responsiveness to environmental change. PAS domains are multifunctional structural motifs that allow protein-protein interactions amongst family members, typically forming heterodimeric transcription factors to affect the transcription of target genes. Prototypical PAS domain-dependent pathways include the circadian clock network and metabolic regulation of the xenobiotic response through the aryl hydrocarbon receptor (AhR). Both pathways are increasingly linked to health, and alteration in their function contributes to development of disease. The AhR demonstrates promiscuity in ligand binding and selectivity during heterodimer formation, which allows varied combinations of protein-protein interactions with other Per-Arnt-Sim (PAS) domain-containing proteins and crosstalk amongst signalling pathways, including the molecular clockworks. AhR and the circadian signalling pathways are highly integrated and reciprocally regulated. AhR exhibits a rhythmic expression and time-dependent sensitivity to activation by AhR agonists. Conversely, AhR influences amplitude and phase of rhythms in circadian clock genes, hormones, and behaviour. Understanding the molecular interactions between AhR and the clock provides insight into physiological regulation of rhythmic processes and provides an innovative approach to development of therapeutics.

Aryl hydrocarbon receptor deficiency protects mice from diet-induced adiposity and metabolic disorders through increased energy expenditure.

BACKGROUND/OBJECTIVES: Epidemics of obesity and diabetes are escalating. High-calorie/high-fat food is a major cause for these global health issues, but molecular mechanisms underlying high-fat, diet-induced obesity are still not well understood. The aryl hydrocarbon receptor (AhR), a transcription factor that acts as a xenobiotic sensor, mediates environmental toxicant-induced obesity, insulin resistance and development of diabetes. AhR also influences lipid metabolism and diet-induced obesity. The effects of AhR deficiency on diet-induced obesity, hepatic steatosis and insulin resistance were examined. METHODS: Male wild-type (WT), AhR null (AhR(-/-)) and AhR heterozygote (AhR(+/-)) mice were fed a normal chow diet (NCD, 10% kcal from fat) or a high-fat diet (HFD, 60% kcal from fat) for up to 14 weeks. Adiposity, adipose and liver morphology, insulin signaling, metabolic parameters and gene profiles were assessed. RESULTS: AhR deficiency protected against HFD-induced obesity, hepatic steatosis, insulin resistance and inflammation. Moreover, AhR deficiency preserved insulin signaling in major metabolic tissues. These protective effects result from a higher energy expenditure in AhR-deficient mice compared with WT. Levels of transcript for both the thermogenic gene, uncoupling protein 1 (Ucp1), in brown adipose tissue and mitochondrial ß-oxidation genes in muscle were significantly higher in AhR(-/-) and AhR(+/-) mice compared with WT. CONCLUSIONS: This work documents a physiologically relevant function for AhR in regulation of body weight, hepatic fat deposition, insulin sensitivity and energy expenditure under HFD exposure, suggesting that AhR signaling may be developed as a potential therapeutic target for treatment of obesity and metabolic disorders.

Beta-naphthoflavone (DB06732) mediates estrogen receptor-positive breast cancer cell cycle arrest through AhR-dependent regulation of PI3K/AKT and MAPK/ERK signaling.

Beta-naphthoflavone (BNF, DB06732) is an agonist of aryl hydrocarbon receptor (AhR) and a putative chemotherapeutic agent that has antitumor activity against mammary carcinomas in vivo. However, the mechanism by which BNF exerts this antitumor effect remains unclear. Thus, we explored mechanisms of BNF's antitumor effects in human breast cancer cells. This study showed that BNF suppressed cell proliferation and induced cell cycle arrest in the G0/G1 phase with downregulation of cyclin D1/D3 and CDK4 and upregulation of p21(Cip1/Waf1), leading to a senescence-like phenotype in estrogen receptor (ER)-positive MCF-7 cells, but not in ER-negative MDA-MB-231 cells. In addition, BNF inhibited PI3K/AKT signaling, and the PI3K inhibitor, LY294,002, exhibited the same inhibitory effects on cyclinD1/D3, CDK4 and the cell cycle as BNF. Interestingly, BNF activated mitogen-activated protein kinase-extracellular signal-regulated kinase (MAPK-ERK) signaling, and more notably, MEK inhibitor PD98059 significantly blocked the BNF-induced cell cycle arrest and upregulation of p21(Cip1/Waf1). Furthermore, specific ERα and AhR siRNA studies indicate that ERα is required in BNF-induced p21(Cip1/Waf1) expression, and BNF-mediated cell cycle arrest and modulation of AKT and ERK signaling is AhR-dependent. Taken together, AhR-dependent inhibition of the PI3K/AKT pathway, activation of MAPK/ERK and modulation of ERα is a novel mechanism underlying BNF-mediated antitumor effects in breast cancer, which may represent a promising strategy to be exploited in future clinical trials.

Interplay between Dioxin-mediated signaling and circadian clock: a possible determinant in metabolic homeostasis.

The rotation of the earth on its axis creates the environment of a 24 h solar day, which organisms on earth have used to their evolutionary advantage by integrating this timing information into their genetic make-up in the form of a circadian clock. This intrinsic molecular clock is pivotal for maintenance of synchronized homeostasis between the individual organism and the external environment to allow coordinated rhythmic physiological and behavioral function. Aryl hydrocarbon receptor (AhR) is a master regulator of dioxin-mediated toxic effects, and is, therefore, critical in maintaining adaptive responses through regulating the expression of phase I/II drug metabolism enzymes. AhR expression is robustly rhythmic, and physiological cross-talk between AhR signaling and circadian rhythms has been established. Increasing evidence raises a compelling argument that disruption of endogenous circadian rhythms contributes to the development of disease, including sleep disorders, metabolic disorders and cancers. Similarly, exposure to environmental pollutants through air, water and food, is increasingly cited as contributory to these same problems. Thus, a better understanding of interactions between AhR signaling and the circadian clock regulatory network can provide critical new insights into environmentally regulated disease processes. This review highlights recent advances in the understanding of the reciprocal interactions between dioxin-mediated AhR signaling and the circadian clock including how these pathways relate to health and disease, with emphasis on the control of metabolic function.

Sex-Dependent Effects of Chronic Circadian Disruption in AßPP/PS1 Mice.

BACKGROUND: Chronic disruption of the circadian timing system, often reflected as a loss of restful sleep, also includes myriad other pathophysiological effects. OBJECTIVE: The current study examined how chronic circadian disruption (CD) could contribute to pathology and rate of progression in the AßPP/PS1 mouse model of Alzheimer's disease (AD). METHODS: A chronic CD was imposed until animals reached 6 or 12 months of age in AßPP/PS1 and C57BL/6J control mice. Home cage activity was monitored for a period of 3-4 weeks prior to the endpoint along with a single timepoint measure of glucose sensitivity. To assess long term effects of CD on the AD phenotype, animals were re-entrained to a no disruption (ND) schedule just prior to the endpoint, after which a Morris water maze (MWM) was used to assess spatial learning and memory. RESULTS: Dampening of nighttime activity levels occurred in disrupted animals, and female animals demonstrated a greater adaptability to CD. Diminished arginine vasopressin (AVP) and vasoactive intestinal peptide (VIP) levels in the suprachiasmatic nucleus (SCN) of 12-month male AßPP/PS1 exposed to the CD paradigm were observed, potentially accounting for the diminished re-entrainment response. Similarly, CD worsened performance in the MWM in 12-month male AßPP/PS1 animals, whereas no effect was seen in females. CONCLUSIONS: Collectively, these findings show that exposure to chronic CD impairs circadian behavioral patterns and cognitive phenotypes of AßPP/PS1 mouse model in a sex-dependent manner.

Deficiency of Adipose Aryl Hydrocarbon Receptor Protects against Diet-Induced Metabolic Dysfunction through Sexually Dimorphic Mechanisms.

The molecular mechanisms underlying diet-induced obesity are complex and remain unclear. The activation of the aryl hydrocarbon receptor (AhR), a xenobiotic sensor, by obesogens may contribute to diet-induced obesity through influences on lipid metabolism and insulin resistance acting at various sites, including adipose tissue. Thus, our hypothesis was that conditional AhR depletion, specifically from mature adipose tissue (CadKO), would improve high-fat diet (HFD)-induced metabolic dysfunction. CadKO protects mice from HFD-induced weight gain. CadKO females eat fewer calories, leading to increased energy expenditure (EE) and improved glucose tolerance on HFD. Our exploration of adipose tissue biology suggests that the depletion of AhR from adipocytes provides female mice with an increased capacity for adipogenesis and lipolysis, allowing for the maintenance of a healthy adipocyte phenotype. The HFD-induced leptin rise was reduced in CadKO females, but the hypothalamic leptin receptor (LepR) was increased in the energy regulatory regions of the hypothalamus, suggesting an increased sensitivity to leptin. The estrogen receptor α (ERα) was higher in CadKO female adipose tissue and the hypothalamus. CadKO males displayed a delayed progression of obesity and insulin resistance. In males, CadKO ameliorated proinflammatory adipocytokine secretion (such as TNFα, IL1ß, IL6) and displayed reduced inflammatory macrophage infiltration into adipose depots. Overall, CadKO improves weight control and systemic glucose homeostasis under HFD challenge but to a more profound extent in females. CadKO facilitates a lean phenotype in females and mediates healthy adipose-hypothalamic crosstalk. In males, adipose-specific AhR depletion delays the development of obesity and insulin resistance through the maintenance of healthy crosstalk between adipocytes and immune cells.

Aryl hydrocarbon receptor affects circadian-regulated lipolysis through an E-Box-dependent mechanism.

An internal circadian clock regulates timing of systemic energy homeostasis. The central clock in the hypothalamic suprachiasmatic nucleus (SCN) directs local clocks in peripheral tissues such as liver, muscle, and adipose tissue to synchronize metabolism with food intake and rest/activity cycles. Aryl hydrocarbon receptor (AhR) interacts with the molecular circadian clockworks. Activation of AhR dampens rhythmic expression of core clock genes, which may lead to metabolic dysfunction. Given the importance of appropriately-timed adipose tissue function to regulation of energy homeostasis, this study focused on mechanisms by which AhR may influence clock-controlled adipose tissue activity. We hypothesized that AhR activation in adipose tissue would impair lipolysis by dampening adipose rhythms, leading to a decreased lipolysis rate during fasting, and subsequently, altered serum glucose concentrations. Levels of clock gene and lipolysis gene transcripts in mouse mesenchymal stem cells (BMSCs) differentiated into mature adipocytes were suppressed by the AhR agonist ß-napthoflavone (BNF), in an AhR dependent manner. BNF altered rhythms of core clock gene and lipolysis gene transcripts in C57bl6/J mice. BNF reduced serum free fatty acids, glycerol and liver glycogen. Chromatin immunoprecipitation indicated that BNF increased binding of AhR to E-Box elements in clock gene and lipolysis gene promoters. These data establish a link between AhR activation and impaired lipolysis, specifically by altering adipose tissue rhythmicity. In response to the decreased available energy from impaired lipolysis, the body increases glycogenolysis, thereby degrading more glycogen to provide necessary energy.

Assessing Sex-Specific Circadian, Metabolic, and Cognitive Phenotypes in the AßPP/PS1 and APPNL-F/NL-F Models of Alzheimer's Disease.

BACKGROUND: Circadian disruption has long been recognized as a symptom of Alzheimer's disease (AD); however, emerging data suggests that circadian dysfunction occurs early on in disease development, potentially preceding any noticeable cognitive deficits. OBJECTIVE: This study compares the onset of AD in male and female wild type (C57BL6/J), transgenic (AßPP/PS1), and knock-in (APPNL-F/NL-F) AD mouse models from the period of plaque initiation (6 months) through 12 months. METHODS: Rhythmic daily activity patterns, glucose sensitivity, cognitive function (Morris water maze, MWM), and AD pathology (plaques formation) were assessed. A comparison was made across sexes. RESULTS: Sex-dependent hyperactivity in AßPP/PS1 mice was observed. In comparison to C57BL/6J animals, 6-month-old male AßPP/PS1 demonstrated nighttime hyperactivity, as did 12-month-old females. Female AßPP/PS1 animals performed significantly worse on a MWM task than AßPP/PS1 males at 12 months and trended toward increased plaque pathology. APPNL-F/NL-F 12-month-old males performed significantly worse on the MWM task compared to 12-month-old females. Significantly greater plaque pathology occurred in AßPP/PS1 animals as compared to APPNL-F/NL-F animals. Female AßPP/PS1 animals performed significantly worse than APPNL-F/NL-F animals in spatial learning and memory tasks, though this was reversed in males. CONCLUSION: Taken together, this study provides novel insights into baseline sex differences, as well as characterizes baseline diurnal activity variations, in the AßPP/PS1 and APPNL-F/NL-F AD mouse models.

The Role of AhR in the Hallmarks of Brain Aging: Friend and Foe.

In recent years, aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, has been considered to be involved in aging phenotypes across several species. This receptor is a highly conserved biosensor that is activated by numerous exogenous and endogenous molecules, including microbiota metabolites, to mediate several physiological and toxicological functions. Brain aging hallmarks, which include glial cell activation and inflammation, increased oxidative stress, mitochondrial dysfunction, and cellular senescence, increase the vulnerability of humans to various neurodegenerative diseases. Interestingly, many studies have implicated AhR signaling pathways in the aging process and longevity across several species. This review provides an overview of the impact of AhR pathways on various aging hallmarks in the brain and the implications for AhR signaling as a mechanism in regulating aging-related diseases of the brain. We also explore how the nature of AhR ligands determines the outcomes of several signaling pathways in brain aging processes.

Reproducibility of adipogenic responses to metabolism disrupting chemicals in the 3T3-L1 pre-adipocyte model system: An interlaboratory study.

The 3T3-L1 murine pre-adipocyte line is an established cell culture model for screening Metabolism Disrupting Chemicals (MDCs). Despite a need to accurately identify MDCs for further evaluation, relatively little research has been performed to comprehensively evaluate reproducibility across laboratories, assess factors that might contribute to varying degrees of differentiation between laboratories (media additives, plastics, cell source, etc.), or to standardize protocols. As such, the goals of this study were to assess interlaboratory variability of efficacy and potency outcomes for triglyceride accumulation and pre-adipocyte proliferation using the mouse 3T3-L1 pre-adipocyte cell assay to test chemicals. Ten laboratories from five different countries participated. Each laboratory evaluated one reference chemical (rosiglitazone) and three blinded test chemicals (tributyltin chloride, pyraclostrobin, and bisphenol A) using: 1) their Laboratory-specific 3T3-L1 Cells (LC) and their Laboratory-specific differentiation Protocol (LP), 2) Shared 3T3-L1 Cells (SC) with LP, 3) LC with a Shared differentiation Protocol (SP), and 4) SC with SP. Blinded test chemical responses were analyzed by the coordinating laboratory. The magnitude and range of bioactivities reported varied considerably across laboratories and test conditions, though the presence or absence of activity for each tested chemical was more consistent. Triglyceride accumulation activity determinations for rosiglitazone ranged from 90 to 100% across test conditions, but 30-70 % for pre-adipocyte proliferation; this was 40-80 % for triglyceride accumulation induced by pyraclostrobin, 80-100 % for tributyltin, and 80-100 % for bisphenol A. Consistency was much lower for pre-adipocyte proliferation, with 30-70 % active determinations for pyraclostrobin, 30-50 % for tributyltin, and 20-40 % for bisphenol A. Greater consistency was observed for the SC/SP assessment. As such, working to develop a standardized adipogenic differentiation protocol represents the best strategy for improving consistency of adipogenic responses using the 3T3-L1 model to reproducibly identify MDCs and increase confidence in reported outcomes.

In vivo circadian rhythms in gonadotropin-releasing hormone neurons.

Although it is generally accepted that the circadian clock provides a timing signal for the luteinizing hormone (LH) surge, mechanistic explanations of this phenomenon remain underexplored. It is known, for example, that circadian locomotor output cycles kaput (clock) mutant mice have severely dampened LH surges, but whether this phenotype derives from a loss of circadian rhythmicity in the suprachiasmatic nucleus (SCN) or altered circadian function in gonadotropin-releasing hormone (GnRH) neurons has not been resolved. GnRH neurons can be stimulated to cycle with a circadian period in vitro and disruption of that cycle disturbs secretion of the GnRH decapeptide. We show that both period-2 (PER2) and brain muscle Arnt-like-1 (BMAL1) proteins cycle with a circadian period in the GnRH population in vivo. PER2 and BMAL1 expression both oscillate with a 24-hour period, with PER2 peaking during the night and BMAL1 peaking during the day. The population, however, is not as homogeneous as other oscillatory tissues with only about 50% of the population sharing peak expression levels of BMAL1 at zeitgeber time 4 (ZT4) and PER2 at ZT16. Further, a light pulse that induced a phase delay in the activity rhythm of the GnRH-eGFP mice caused a similar delay in peak expression levels of BMAL1 and PER2. These studies provide direct evidence for a functional circadian clock in native GnRH neurons with a phase that closely follows that of the SCN.

Behavioral rhythmicity of mice lacking AhR and attenuation of light-induced phase shift by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Transcription factors belonging to the Per/Arnt/Sim (PAS) domain family are highly conserved and many are involved in circadian rhythm regulation. One member of this family, aryl hydrocarbon receptor (AhR), is an orphan receptor whose physiological role is unknown. Recent findings have led to the hypothesis that AhR has a role in circadian rhythm, which is the focus of the present investigation. First, time-of-day-dependent mRNA expression of AhR and its signaling target, cytochrome p4501A1 (Cyp1a1), was determined in C57BL/6J mice by quantitative RT-PCR. Circadian expression of AhR and Cyp1a1 was observed both in the suprachiasmatic nucleus (SCN) and liver. Next, the circadian phenotype of mice lacking AhR (AhRKO) was investigated using behavioral monitoring. Intact AhRKO mice had robust circadian rhythmicity with a similar tau under constant conditions compared to wild-type mice, but a significant difference in tau was observed between genotypes in ovariectomized female mice. Time to reentrainment following 6-h advances or delays of the light/dark cycle was not significantly different between genotypes. However, mice exposed to the AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 1 microg/kg of body weight) displayed decreased phase shifts in response to light and had altered expression of Per1 and Bmal1. These results suggest that chronic activation of AhR may affect the ability of the circadian timekeeping system to adjust to alterations in environmental lighting by affecting canonical clock genes. Further studies are necessary to decipher the mechanism of how AhR agonists could disrupt light-induced phase shifts. If AhR does have a role in circadian rhythm, it may share redundant roles with other PAS domain proteins and/or the role of AhR may not be exhibited in the behavioral activity rhythm, but could be important elsewhere in the peripheral circadian system.

Protein kinase G type II is required for night-to-day progression of the mammalian circadian clock.

Circadian clocks comprise a cyclic series of dynamic cellular states, characterized by the changing availability of substrates that alter clock time when activated. To determine whether circadian clocks, like the cell cycle, exhibit regulation by key phosphorylation events, we examined endogenous kinase regulation of timekeeping in the mammalian suprachiasmatic nucleus (SCN). Short-term inhibition of PKG-II but not PKG-Ibeta using antisense oligodeoxynucleotides delayed rhythms of electrical activity and Bmal1 mRNA. Phase resetting was rapid and dynamic; inhibition of PKG-II forced repetition of the last 3.5 hr of the cycle. Chronic inhibition of PKG-II disrupted electrical activity rhythms and tonically increased Bmal1 mRNA. PKG-II-like immunoreactivity was detected after coimmunoprecipitation with CLOCK, and CLOCK was phosphorylated in the presence of active PKG-II. PKG-II activation may define a critical control point for temporal progression into the daytime domain by acting on the positive arm of the transcriptional/translational feedback loop.

Effects of tryptophan photoproducts in the circadian timing system: searching for a physiological role for aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AhR) mediates adverse effects of dioxins, but its physiological role remains ambiguous. The similarity between AhR and canonical circadian clock genes suggests potential involvement of AhR in regulation of circadian timing. Photoproducts of tryptophan (TRP), including 6-formylindolo[3,2-b]carbazole (FICZ), have high affinity for AhR and are postulated as endogenous ligands. Although TRP photoproducts activate AhR signaling in vitro, their effects in vivo have not been investigated in mammals. Because TRP photoproducts may act as transducers of light, we examined their effects on the circadian clock. Intraperitoneal injection of TRP photoproducts or FICZ to C57BL/6J mice dose dependently induced AhR downstream targets, cytochrome P4501A1 (CYP1A1) and cytochrome P4501B1 mRNA expression, in liver. c-fos mRNA, a commonly used marker for light responses, was also induced with FICZ, and all responses were AhR dependent. A rat-immortalized suprachiasmatic nucleus (SCN) cell line, SCN 2.2, was used to examine the direct effect of TRP photoproducts on the molecular clock. Both TRP photoproducts and FICZ-increased CYP1A1 expression and prolonged FICZ incubation altered the circadian expression of clock genes (Per1, Cry1, and Cry2) in SCN 2.2 cells. Furthermore, FICZ inhibited glutamate-induced phase shifting of the mouse SCN electrical activity rhythm. Circadian light entrainment is critical for adjustment of the endogenous rhythm to environmental light cycle. Our results reveal a potential for TRP photoproducts to modulate light-dependent regulation of circadian rhythm through triggering of AhR signaling. This may lead to further understanding of toxicity of dioxins and the role of AhR in circadian rhythmicity.

Aryl hydrocarbon receptor-deficient mice are protected from high fat diet-induced changes in metabolic rhythms.

High fat diet (HFD) consumption alters the synchronized circadian timing system resulting in harmful loss, gain or shift of transcriptional oscillations. The aryl hydrocarbon receptor (AhR) shares structural homology to clock genes, containing both PAS domains and basic helix-loop helix structural motifs, allowing for interaction with components of the primary circadian feedback loop. Activation of AhR alters circadian rhythmicity, primarily through inhibition of Clock/Bmal1-mediated regulation of Per1. AhR-deficient mice are protected from diet-induced metabolic dysfunction, exhibiting enhanced insulin sensitivity and glucose tolerance. This study examined whether AhR haploinsufficiency can also protect against diet-induced alterations in rhythm. After feeding AhR+/+ and AhR+/- mice an HFD (60% fat) for 15 weeks, samples were collected every 4 hours over a 24-hour period. HFD altered the rhythm of serum glucose and the metabolic transcriptome, including hepatic nuclear receptors Rev-erbα and PPARγ in wild-type c57bl6/j mice. AhR reduction provided protection against diet-induced transcriptional oscillation changes; serum glucose and metabolic gene rhythms were protected from the disruption caused by HFD feeding. These data highlight the critical role of AhR signaling in the regulation of metabolism and provide a potential therapeutic target for diseases characterized by rhythmic desynchrony.

Aryl Hydrocarbon Receptor Deficiency Alters Circadian and Metabolic Rhythmicity.

PAS domain-containing proteins can act as environmental sensors that capture external stimuli to allow coordination of organismal physiology with the outside world. These proteins permit diverse ligand binding and heterodimeric partnership, allowing for varied combinations of PAS-dependent protein-protein interactions and promoting crosstalk among signaling pathways. Previous studies report crosstalk between circadian clock proteins and the aryl hydrocarbon receptor (AhR). Activated AhR forms a heterodimer with the circadian clock protein Bmal1 and thereby functionally inhibits CLOCK/Bmal1 activity. If physiological activation of AhR through naturally occurring, endogenous ligands inhibits clock function, it seems plausible to hypothesize that decreased AhR expression releases AhR-induced inhibition of circadian rhythms. Because both AhR and the clock are important regulators of glucose metabolism, it follows that decreased AhR will also alter metabolic function. To test this hypothesis, rhythms of behavior, metabolic outputs, and circadian and metabolic gene expression were measured in AhR-deficient mice. Genetic depletion of AhR enhanced behavioral responses to changes in the light-dark cycle, increased rhythmic amplitude of circadian clock genes in the liver, and altered rhythms of glucose and insulin. This study provides evidence of AhR-induced inhibition that influences circadian rhythm amplitude.

Role of Aryl Hydrocarbon Receptor in Circadian Clock Disruption and Metabolic Dysfunction.

The prevalence of metabolic syndrome, a clustering of three or more risk factors that include abdominal obesity, increased blood pressure, and high levels of glucose, triglycerides, and high-density lipoproteins, has reached dangerous and costly levels worldwide. Increases in morbidity and mortality result from a combination of factors that promote altered glucose metabolism, insulin resistance, and metabolic dysfunction. Although diet and exercise are commonly touted as important determinants in the development of metabolic dysfunction, other environmental factors, including circadian clock disruption and activation of the aryl hydrocarbon receptor (AhR) by dietary or other environmental sources, must also be considered. AhR binds a range of ligands, which prompts protein-protein interactions with other Per-Arnt-Sim (PAS)-domain-containing proteins and subsequent transcriptional activity. This review focuses on the reciprocal crosstalk between the activated AhR and the molecular circadian clock. AhR exhibits a rhythmic expression and time-dependent sensitivity to activation by AhR agonists. Conversely, AhR activation influences the amplitude and phase of expression of circadian clock genes, hormones, and the behavioral responses of the clock system to changes in environmental illumination. Both the clock and AhR status and activation play significant and underappreciated roles in metabolic homeostasis. This review highlights the state of knowledge regarding how AhR may act together with the circadian clock to influence energy metabolism. Understanding the variety of AhR-dependent mechanisms, including its interactions with the circadian timing system that promote metabolic dysfunction, reveals new targets of interest for maintenance of healthy metabolism.

Circadian Disruption Reveals a Correlation of an Oxidative GSH/GSSG Redox Shift with Learning and Impaired Memory in an Alzheimer's Disease Mouse Model.

It is unclear whether pre-symptomatic Alzheimer's disease (AD) causes circadian disruption or whether circadian disruption accelerates AD pathogenesis. In order to examine the sensitivity of learning and memory to circadian disruption, we altered normal lighting phases by an 8 h shortening of the dark period every 3 days (jet lag) in the APPSwDI NOS2-/- model of AD (AD-Tg) at a young age (4-5 months), when memory is not yet affected compared to non-transgenic (non-Tg) mice. Analysis of activity in 12-12 h lighting or constant darkness showed only minor differences between AD-Tg and non-Tg mice. Jet lag greatly reduced activity in both genotypes during the normal dark time. Learning on the Morris water maze was significantly impaired only in the AD-Tg mice exposed to jet lag. However, memory 3 days after training was impaired in both genotypes. Jet lag caused a decrease of glutathione (GSH) levels that tended to be more pronounced in AD-Tg than in non-Tg brains and an associated increase in NADH levels in both genotypes. Lower brain GSH levels after jet lag correlated with poor performance on the maze. These data indicate that the combination of the environmental stress of circadian disruption together with latent stress of the mutant amyloid and NOS2 knockout contributes to cognitive deficits that correlate with lower GSH levels.

Circadian clock-controlled regulation of cGMP-protein kinase G in the nocturnal domain.

The suprachiasmatic nucleus (SCN) circadian clock exhibits a recurrent series of dynamic cellular states, characterized by the ability of exogenous signals to activate defined kinases that alter clock time. To explore potential relationships between kinase activation by exogenous signals and endogenous control mechanisms, we examined clock-controlled protein kinase G (PKG) regulation in the mammalian SCN. Signaling via the cGMP-PKG pathway is required for light- or glutamate (GLU)-induced phase advance in late night. Spontaneous cGMP-PKG activation occurred at the end of subjective night in free-running SCN in vitro. Phasing of the SCN rhythm in vitro was delayed by approximately 3 hr after treatment with guanylyl cyclase (GC) inhibitors, PKG inhibition, or antisense oligodeoxynucleotide (alphaODN) specific for PKG, but not PKA inhibitor or mismatched ODN. This sensitivity to GC-PKG inhibition was limited to the same 2 hr time window demarcated by clock-controlled activation of cGMP-PKG. Inhibition of the cGMP-PKG pathway at this time caused delays in the phasing of four endogenous rhythms: wheel-running activity, neuronal activity, cGMP, and Per1. Timing of the cGMP-PKG-necessary window in both rat and mouse depended on clock phase, established by the antecedent light/dark cycle rather than solar time. Because behavioral, neurophysiological, biochemical, and molecular rhythms showed the same temporal sensitivities and qualitative responses, we predict that clock-regulated GC-cGMP-PKG activation may provide a necessary cue as to clock state at the end of the nocturnal domain. Because sensitivity to phase advance by light-GLU-activated GC-cGMP-PKG occurs in juxtaposition, these signals may induce a premature shift to this PKG-necessary clock state.