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.

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-ß-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 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.

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.

Considerations for the use of anesthetics in neurotoxicity studies.

Anesthetics are widely used in experiments investigating neurotoxicity and neuroprotection; however, these agents are known to interfere with the outcome of these experiments. The purpose of this overview is to review these effects and suggest methods for minimizing unintended consequences on experimental outcomes. Information on the neuroprotective and neurotoxic effects of isoflurane, dexmedetomidine, propofol, ketamine, barbiturates, halothane, xenon, carbon dioxide, and nitrous oxide is summarized. The pertinent cell signaling pathways of these agents are discussed. Methods of humane animal euthanasia without anesthetics are considered. Most anesthetics alter the processes of neuronal survival and death. When designing survival surgeries, sham controls subjected to anesthesia but not the surgical intervention should be compared with controls subjected to neither anesthesia nor surgery. Additional controls could include using an anesthetic with a different mechanism of action from the primary anesthetic used. Because the effects of anesthetics lessen with time after surgery, survival surgeries should include later time points until at least 7 d after the procedure. Humane methods of animal euthanasia that do not require anesthetics exist and should be used whenever appropriate.

Time-of-day affects expression of hippocampal markers for ischemic damage induced by global ischemia.

Time-of-day-dependent variation in neuronal ischemia is well documented. Whether this results from changes in time-of-day variation in susceptibility or from other causative factors remains unclear. We hypothesize that hippocampal cells exhibit variation in activation of cell death predictive markers in response to ischemia induced at different times-of-day. Changes in hippocampal circadian clock gene rhythmicity may also be associated with ischemia. Transient global ischemia was induced in rats at three times of day and animals were sacrificed 24 h later. Hippocampal caspase-3, -8 and -9 transcripts and active proteins and calbindin protein were measured in the CA1 region of the hippocampus. In a second study, 24-h rhythms of circadian regulatory transcripts were determined in hippocampus after global ischemia. Caspase-3, -8 and -9 transcripts and active protein levels were increased substantially when ischemia occurred in early night (ZT14); smaller changes were observed in late night (ZT20, or day ZT6). Calbindin levels decreased following ischemia, especially at ZT14. Ischemia shifted the rhythm of the Per1 transcript; peak expression occurred 6 h earlier following ischemia. Rhythms of Cry1 and Bmal1 were not altered. Greater induction of caspases and decline of calbindin when ischemia was performed at ZT14 than at ZT20 or ZT6 support the concept of increased hippocampal susceptibility to ischemia at ZT14. Alteration of the Per1 transcript suggests a potential role for the circadian clock in this process. Notably, ZT14 represents the beginning of the rats' nocturnal period of activity, corresponding to the time when humans experience the greatest neuronal ischemic damage from stroke.

Suppression of catatonia-like signs by lorazepam in nonconvulsive status epilepticus without seizure termination.

Nonconvulsive status epilepticus (NCSE) cannot be reliably distinguished from catatonia by signs or symptoms. The authors report on a 68-year-old man with endogenous major depression who displayed catatonia-like psychopathology, which temporarily disappeared with lorazepam. An EEG then revealed NCSE. Presumably, lorazepam suppressed seizure in areas where it had caused clouding of consciousness, but it did not suppress all seizure activity. When lorazepam was stopped, the catatonia-like delirium returned; it disappeared again with valproate administration. This case illustrates that the reduction of signs consistent with catatonia by benzodiazepines does not by itself confirm catatonia, even in patients with endogenous depression.