Stacey L. Krager

Aryl Hydrocarbon Receptor Deficiency Enhances Insulin Sensitivity and Reduces PPAR-α Pathway Activity in Mice

Background: Numerous man-made pollutants activate the aryl hydrocarbon receptor (AhR) and are risk factors for type 2 diabetes. AhR signaling also affects molecular clock genes to influence glucose metabolism. Objective: We investigated mechanisms by which AhR activation affects glucose metabolism. Methods: Glucose tolerance, insulin resistance, and expression of peroxisome proliferator–activated receptor-α (PPAR-α) and genes affecting glucose metabolism or fatty acid oxidation and clock gene rhythms were investigated in wild-type (WT) and AhR-deficient [knockout (KO)] mice. AhR agonists and small interfering RNA (siRNA) were used to examine the effect of AhR on PPAR-α expression and glycolysis in the liver cell line Hepa-1c1c7 (c7) and its c12 and c4 derivatives. Brain, muscle ARNT-like protein 1 (Bmal1) siRNA and Ahr or Bmal1 expression plasmids were used to analyze the effect of BMAL1 on PPAR-α expression in c7 cells. Results: KO mice displayed enhanced insulin sensitivity and improved glucose tolerance, accompanied by decreased PPAR-α and key gluconeogenic and fatty acid oxidation enzymes. AhR agonists increased PPAR-α expression in c7 cells. Both Ahr and Bmal1 siRNA reduced PPAR-α and metabolism genes. Moreover, rhythms of BMAL1 and blood glucose were altered in KO mice. Conclusions: These results indicate a link between AhR signaling, circadian rhythms, and glucose metabolism. Furthermore, hepatic activation of the PPAR-α pathway provides a mechanism underlying AhR-mediated insulin resistance.

Disruption of Clock/BMAL1 Transcriptional Activity is Responsible for Aryl Hydrocarbon Receptor-Mediated Regulation of Period1 Gene

The aryl hydrocarbon receptor (AhR) is a period-aryl hydrocarbon receptor nuclear transporter-simple minded domain transcription factor that shares structural similarity with circadian clock genes and readily interacts with components of the molecular clock. Activation of AhR by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters behavioral circadian rhythms and represses the Period1 (Per1) gene in murine hematopoietic stem and progenitor cells. Per1 expression is driven by circadian locomotor activity cycles kaput-brain muscle ARNT-like (CLOCK-BMAL1)-dependent activation of Eboxes in the Per1 promoter. We hypothesized that the effects of AhR activation on the circadian clock are mediated by disruption of CLOCK-BMAL1 function and subsequent Per1 gene suppression. Effects of AhR activation on rhythmic Per1 transcripts were examined in livers of mice after treatment with the AhR agonist, TCDD; the molecular mechanisms of Per1 repression by AhR were determined in hepatoma cells using TCDD and beta-napthoflavone as AhR activators. This study reports, for the first time, that AhR activation by TCDD alters the Per1 rhythm in the mouse liver and that Per1 gene suppression depends upon the presence of AhR. Furthermore, AhR interaction with BMAL1 attenuates CLOCK-BMAL1 activity and decreases CLOCK binding at Ebox1 and Ebox3 in the Per1 promoter. Taken together, these data suggest that AhR activation represses Per1 through disrupting CLOCK-BMAL1 activity, producing dysregulation of rhythmic Per1 gene expression. These data define alteration of the Per1 rhythm as novel signaling events downstream of AhR activation. Downregulation of Per1 could contribute to metabolic disease, cancer, and other detrimental effects resulting from exposure to certain environmental pollutants.

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 β-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.

CIRCADIAN CLOCK DISRUPTION IN THE MOUSE OVARY IN RESPONSE TO 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN

Activation of the aryl hydrocarbon receptor (AhR) by the highly toxic, prototypical ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or other dioxin-like compounds compromises ovarian function by altering follicle maturation and steroid synthesis. Although alteration of transcription after nuclear translocation and heterodimerization of AhR with its binding partner, aryl hydrocarbon nuclear transporter (ARNT), is often cited as a primary mechanism for mediating the toxic effects of dioxins, recent evidence indicates that crosstalk between AhR and several other signaling pathways also occurs. Like the circadian clock genes, AhR is a member of the basic helix-loop-helix, Per-ARNT-SIM (bHLH-PAS) domain family of proteins. Thus, these studies tested the hypothesis that TCDD can act to alter circadian clock regulation in the ovary. Adult female c57bl6/J mice entrained to a typical 12h light/12h dark cycle were exposed to a single 1 μg/kg dose of TCDD by gavage. Six days after exposure, animals were released into constant darkness and ovaries were collected every 4h over a 24h period. Quantitative real-time PCR and immunoblot analysis demonstrated that TCDD exposure alters expression of the canonical clock genes, Bmal1 and Per2 in the ovary. AhR transcript and protein, which displayed a circadian pattern of expression in the ovaries of control mice, were also altered after TCDD treatment. Immunohistochemistry studies revealed co-localization of AhR with BMAL1 in various ovarian cell types. Furthermore, co-immunoprecipitation demonstrated time-of-day dependent interactions of AhR with BMAL1 that were enhanced after TCDD treatment. Collectively these studies suggest that crosstalk between classical AhR signaling and the molecular circadian clockworks may be responsible for altered ovarian function after TCDD exposure.

ERK/MAPK Is Essential for Endogenous Neuroprotection in SCN2.2 Cells

Background Glutamate (Glu) is essential to central nervous system function; however excessive Glu release leads to neurodegenerative disease. Strategies to protect neurons are underdeveloped, in part due to a limited understanding of natural neuroprotective mechanisms, such as those present in the suprachiasmatic nucleus (SCN). This study tests the hypothesis that activation of ERK/MAPK provides essential protection to the SCN after exposure to excessive Glu using the SCN2.2 cells as a model. Methodology Immortalized SCN2.2 cells (derived from SCN) and GT1-7 cells (neurons from the neighboring hypothalamus) were treated with 10 mM Glu in the presence or absence of the ERK/MAPK inhibitor PD98059. Cell death was assessed by Live/Dead assay, MTS assay and TUNEL. Caspase 3 activity was also measured. Activation of MAPK family members was determined by immunoblot. Bcl2, neuritin and Bid mRNA (by quantitative-PCR) and protein levels (by immunoblot) were also measured. Principal Findings As expected Glu treatment increased caspase 3 activity and cell death in the GT1-7 cells, but Glu alone did not induce cell death or affect caspase 3 activity in the SCN2.2 cells. However, pretreatment with PD98059 increased caspase 3 activity and resulted in cell death after Glu treatment in SCN2.2 cells. This effect was dependent on NMDA receptor activation. Glu treatment in the SCN2.2 cells resulted in sustained activation of the anti-apoptotic pERK/MAPK, without affecting the pro-apoptotic p-p38/MAPK. In contrast, Glu exposure in GT1-7 cells caused an increase in p-p38/MAPK and a decrease in pERK/MAPK. Bcl2-protein increased in SCN2.2 cells following Glu treatment, but not in GT1-7 cells; bid mRNA and cleaved-Bid protein increased in GT1-7, but not SCN2.2 cells. Conclusions Facilitation of ERK activation and inhibition of caspase activation promotes resistance to Glu excitotoxicity in SCN2.2 cells. Significance Further research will explore ERK/MAPK as a key molecule in the prevention of neurodegenerative processes.

The luteinizing hormone surge regulates circadian clock gene expression in the chicken ovary.

The molecular circadian clock mechanism is highly conserved between mammalian and avian species. Avian circadian timing is regulated at multiple oscillatory sites, including the retina, pineal, and hypothalamic suprachiasmatic nucleus (SCN). Based on the authors’ previous studies on the rat ovary, it was hypothesized that ovarian clock timing is regulated by the luteinizing hormone (LH) surge. The authors used the chicken as a model to test this hypothesis, because the timing of the endogenous LH surge is accurately predicted from the time of oviposition. Therefore, tissues can be removed before and after the LH surge, allowing one to determine the effect of LH on specific clock genes. The authors first examined the 24-h expression patterns of the avian circadian clock genes of Bmal1, Cry1, and Per2 in primary oscillatory tissues (hypothalamus and pineal) as well as peripheral tissues (liver and ovary). Second, the authors determined changes in clock gene expression after the endogenous LH surge. Clock genes were rhythmically expressed in each tissue, but LH influenced expression of these clock genes only in the ovary. The data suggest that expression of ovarian circadian clock genes may be influenced by the LH surge in vivo and directly by LH in cultured granulosa cells. LH induced rhythmic expression of Per1 and Bmal1 in arrhythmic, cultured granulosa cells. Furthermore, LH altered the phase and amplitude of clock gene rhythms in serum-shocked granulosa cells. Thus, the LH surge may be a mechanistic link for communicating circadian timing information from the central pacemaker to the ovary. (Author correspondence: stischkau@siumed.edu)

Aryl Hydrocarbon Receptor Activation Attenuates Per1 Gene Induction and Influences Circadian Clock Resetting

Light-stimulated adjustment of the circadian clock is an important adaptive physiological response that allows maintenance of behavioral synchrony with solar time. Our previous studies indicate that the aryl hydrocarbon receptor (AhR) agonist 2,3,7,8- tetrachlorodibenzo-p-dioxin attenuates light-induced phase resetting in early night. However, the mechanism of inhibition remains unclear. In this study, we showed that another potent AhR agonist-β-naphthoflavone (BNF)-significantly decreased light-induced phase shifts in wild-type (WT) mice, whereas AhR knockout mice had an enhanced response to light that was unaffected by BNF. Mechanistically, BNF blocked light induction of the Per1 transcript in suprachiasmatic nucleus and liver in WT mice, and BNF blocked forskolin (FSK)-induced Per1 transcripts in Hepa-1c1c7 (c7) cells. An E-box decoy did not affect BNF inhibition of FSK-induced Per1 transcripts in c7 cells. cAMP-response element (CRE)-dependent induction of Per1 promoter activity in response to FSK in combination with phorbol 12-tetradecanoate 13-acetate was suppressed in cells that expressed high levels of AhR (c7) compared with cells lacking functional AhR activity (c12). In addition, the inhibitory effect of BNF on FSK-induced Per1 was dependent on phosphorylation of JNK. Together, these results suggest that AhR activation inhibits light-induced phase resetting through the activation of JNK, negative regulation of CREs in the Per1 promoter, and suppression of Per1.

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

ABSTRACT 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.