Date of Award

12-1-2014

Degree Name

Doctor of Philosophy

Department

Pharmacology

First Advisor

Tischkau, Shelley

Abstract

The aryl hydrocarbon receptor (AhR), a ligand-dependent transcription factor, plays a crucial role in regulation of xenobiotic metabolism. AhR is also involved in dioxin-induced metabolic disorders and alteration of circadian rhythm. Furthermore, circadian clock disruption and metabolic dysfunction are integrally associated with each other. This study was designed to understand the mechanisms by which AhR contributes to regulation of circadian clocks, fat metabolism and glucose homeostasis. In the first aim, I have tested whether AhR interacts with the core clock gene, brain and muscle AhR nuclear translocator like-1(BMAL1), disrupting circadian locomotor output cycle kaput (CLOCK)/BMAL1 complex activity, and leading to the suppression of period1 gene (Per1) expression rhythm. My studies indicate that AhR activation by its agonists 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and beta-naphoflavone (BNF) disrupts the rhythm and inhibits the expression of Per1 in mouse liver and hepatoma cell lines, respectively. Mechanistically, the disruption of the rhythm and the inhibition of Per1 expression occur secondary to the interaction between AhR and BMAL1, which attenuates transcriptional activity of the core clock complex CLOCK/BMAL1. These results suggest alteration of the circadian clock as a novel signaling event downstream of AhR activation. The integral relationship between the clock and metabolic function further suggest that AhR activation may cause metabolic dysfunction. In the second aim, I have tested whether AhR activation inhibits Per1 gene induction and influences circadian clock resetting through activation of JNK pathway. AhR activation by it agonists TCDD and BNF decreases light-induced phase shifts in the early night and inhibits light-induced Per1 expression in both suprachiasmatic nucleus (SCN) and liver. Inhibition of Per1 induction results from increased phospho-JNK induced by AhR activation. Taken together, activation of AhR disrupts circadian clock resetting which also could cause metabolic dysfunction. In the third aim, I have tested whether AhR deficiency regulates nuclear receptor peroxisome proliferator-activated receptor a; (PPARa) and alters glucose homeostasis. PPARa, a clock-controlled gene (CCG) that acts as a fat metabolism sensor, is important for lipid metabolism and glucose homeostasis. AhR knockout (AhRKO or AhR-/-) mice exhibit enhanced insulin sensitivity and glucose tolerance, accompanied by decreased expression of PPARa, key gluconeogenic genes, glucose-6 phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) and key fatty acid oxidation enzymes, carnitine palmitoyl transferase1b (CPT1b) and acyl-CoA oxidase (ACO). Conversely, AhR agonists increase PPARa; expression in a BMAL1-dependent manner. In addition, AhRKO mice display altered rhythm for clock genes, clock-controlled genes (CCGs) and physiological blood glucose. These data suggest that AhR may modulate the glucose homeostasis through regulation of CCGs such as PPARa and that PPARa may be an important link between the circadian clock and metabolism. In the fourth aim, I have tested the effects of AhR ablation or attenuation in high-fat diet (HFD)-induced obesity, insulin resistance and hepatic steatosis in mice. Recent studies suggest that PPARα expression is elevated with HFD feeding as an adaptive response to attenuate hepatic steatosis, and PPARa deficiency protects against HFD-induced insulin resistance. AhR-/- as well as AhR heterozygous (AhR+/-) mice are protected from the HFD-induced obesity, insulin resistance, hepatic steatosis and show reduced inflammatory cytokine expression. In addition, AhR-/- and AhR+/- mice display protected insulin signaling, a higher adiponectin and a lower leptin and insulin in serum. Food intake and physical activity are not significantly different among WT, AhR-/- and AhR+/- mice with HFD feeding. Indirect calorimetry has demonstrated that the AhR+/- mice have higher oxygen consumption, CO2 production and heat production. In addition, Real-time PCR data show that uncoupling protein 1(Ucp1) is higher in brown adipose tissue which supports the higher heat production; moreover, the muscle gene profile reveals that the fatty acid beta-oxidation genes and mitochondrial respiratory genes are higher in AHR+/- mice which further support higher energy expenditure in these mice. Collectively, these data suggest that AhR signaling could be a potential target for treatment of obesity and type 2 diabetes, and AhR antagonist may be developed into a drug for these metabolic diseases.

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