Obesity, metabolic syndrome, and diabetes represent a major source of morbidity and mortality in the United States and worldwide. Chronic misalignment of an organism’s internal circadian clock with diurnal, cyclic changes in the external environment, prevalent in professions that require shift work, contributes significantly to Type 2 Diabetes development. Experimentally, only short-term models of circadian disruption have been explored. Therefore, the goal of this study was to establish an animal model of chronic circadian disruption, which would more closely mimic the harmful misalignment associated with metabolic syndrome in clinical studies. Moreover, since high fat diet consumption alters circadian behavior and rhythmic gene expression, contributing to the diet-induced phenotype, I hypothesized that chronic circadian disruption interacts with a high fat diet to worsen metabolic syndrome. To investigate circadian misalignment and diet-induced metabolic syndrome, I examined the contribution of the Aryl Hydrocarbon Receptor (AhR). AhR has similar PAS domain containing motifs as circadian clock proteins allowing for protein/protein interactions and crosstalk between AhR signaling and circadian rhythms. Furthermore, AhR activation is implicated in Type 2 Diabetes risk. To examine chronic circadian disruption, male wild-type (WT; C57Bl/6J) and AhR +/- mice were entrained to 12/12-hour light/dark cycles where lights were on from 10pm-10am and off from 10am-10pm. Misalignment was initiated by delaying the time of lights on by 8 hours on Monday. Mice were exposed to the misalignment schedule Monday-Friday then returned to the entrainment schedule Saturday and Sunday to mimic readjustment to society during the weekend. Circadian misaligned mice were exposed to the altered light schedule for 15 weeks and control animals remained on the12/12-hour light/dark cycle. Mice were fed a normal chow diet (10% fat) or a high fat diet (60% fat). Animals were sacrificed and samples were collected at 4-hour intervals on day 2 of the weekend. Exposure to chronic circadian misalignment by light disruption or high fat diet altered circadian rhythms of behavior, metabolic outputs, and expression of circadian clock, clock-controlled nuclear receptor, and lipid metabolism genes. A combination of light misalignment and high fat diet exacerbated the effects of either treatment alone further disrupting behavior, enhancing % body fat and fasting glucose, and dampening circadian clock gene expression. AhR +/- mice also were protected from the metabolic consequences of chronic misalignment and a high fat diet by resistance to altered behavioral and molecular circadian rhythms and disruption of metabolic outputs. With metabolic syndrome and Type 2 Diabetes occurrence on the rise, it is important to understand all contributing factors, including circadian disruption. Differences between chronic circadian misalignment and high fat diet-induced obesity in WT and AhR +/- mice furthers our understanding of the complex mechanisms that underlie Type 2 Diabetes development and advocates the discovery of potential therapeutic targets for the development of novel treatment options.

No description available.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 3 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 6 uppsatser.

Thesis (Ph. D.)--University of Washington, 2008. / Vita. Includes bibliographical references (leaves 186-207).

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2010.

Cultured human lymphocytes may be assayed for aryl hydrocarbon hydroxylase (AHH) in whole cell preparations. The optimum assay conditions are pH 8.5, and 1.5 mM Mg++. The reaction is linear with time and cell number, and is inhibited by CO. Estradiol may inhibit induction of AHH by 3-methylcholanthrene, but is a poor competitor for the enzyme. A Caucasian population was assayed for AHH activity. The distribution was lognormal; no difference was found in cultured cells from males and females or smokers and nonsmokers. Cells from relatives of lung cancer patients showed higher activity. An American Indian population showed no difference from the Caucasian population in enzyme level. No linkage was found between AHH and 16a-hydroxylase.

The main goal of this thesis was to develop new methods and knowledge that will explain and predict species differences in sensitivity to dioxin-like compounds (DLCs) in birds. The important achievements and results obtained from the four experimental chapters of this thesis are summarized as follow: (1) an efficient luciferase reporter gene (LRG) assay was developed for use with 96-well cell culture plates; (2) the results obtained from LRG assay were shown to be highly correlated to available in ovo toxicity data; (3) amino acids at positions 324 and 380 within the aryl hydrocarbon receptor 1 ligand binding domain (AHR1 LBD) were shown to be responsible for reduced Japanese quail (Coturnix japonica) AHR1 activity to induce a dioxin-responsive reporter gene in comparison to chicken (Gallus gallus domesticus), and ring-necked pheasant (Phasianus colchicus) AHR1 in response to different DLCs; (4) AHR1 LBD sequences of 86 avian species were studied and differences at amino acid sites 256, 257, 297, 324, 337 and 380 were identified. It was discovered that only positions 324 and 380 play a role in AHR1 activity to induce a dioxin-responsive gene; (5) in COS-7 cells expressing chicken AHR1, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) are equipotent inducers of the reporter gene and bind with similar affinity to chicken AHR1, however, in the cells expressing pheasant, Japanese quail and common tern (Sterna hirundo) AHR1, PeCDF is a stronger inducer than TCDD. PeCDF also binds with higher affinity to pheasant and quail AHR1 than TCDD. The results of this thesis show that embryo lethal effect of DLCs in avian species can be predicted by use of two new non-lethal methods: (1) the LRG assay and (2) determination of the identity of the amino acids at positions 324 and 380. The findings and methods described in this thesis will be of use for environmental risk assessments of DLCs.

The aryl hydrocarbon receptor (AHR) mediates the toxic effects of halogenated aromatic hydrocarbons (HAHs), including 2,3,7,8-tetrachlorodibenzo-p-dioxin, 2,3,4,7,8-pentachlorodibenzofuran and 2,3,7,8-tetrachlorodibenzofuran. Y322 is believed to play a role in binding-independent activation of AHR by atypical inducers, such as omeprazole. I examined AHR-mediated regulation of and coactivator recruitment to CYP1A1, CYP1B1, HES1 and TiPARP in T-47D and HuH7 cells. All compounds induced expression of each gene in both cell lines, with some temporal differences between the HAHs and omeprazole. Chromatin immunoprecipitation assays demonstrated activator-, cell line- and gene-selectivity in AHR coactivator recruitment. Omeprazole induced AHR degradation which was prevented by MG-132 pre-treatment. Y322 was found to be important for maximal AHR activation by 2,3,7,8-TCDD and 2,3,4,7,8-PeCDF, but required for 2,3,7,8-TCDF and Omp in an AHR-deficient MCF-7 cells. My findings provide further evidence for cell-, gene- and ligand-dependent differences in AHR-mediated gene expression and coactivator recruitment, and a role for Y322 in AHR activation.