Date of Award

12-1-2018

Degree Name

Doctor of Philosophy

Department

Molecular Biology, Microbiology and Biochemistry

First Advisor

Tischkau, Shelley

Abstract

Type 2 diabetes is a metabolic disorder characterized by increased glucose concentrations in the blood due to decreased insulin sensitivity. The worldwide incidence of diabetes has increased remarkably over the last two decades. Obesity, due to increased consumption of calorie dense diets, and sedentary life styles, is commonly cited as a primary cause. However, many epidemiological studies have established a relationship between insulin resistance and exposure to environmental chemicals such as persistent organic pollutants (POPs). The mechanisms by which POPs alter metabolism remain poorly understood, although their lipophilic nature suggests a role in adipose tissue function. The Tischkau lab has established a relationship between Aryl hydrocarbon Receptor (AhR) activation by different types of POPs and increased risk of insulin resistance. This dissertation, therefore, explored the effects of AhR activation by POPs on adipose tissue function. Adipose tissue regulates systemic glucose and lipid metabolism through production of hormones and cytokines that regulate appetite and energy homeostasis. It is well-known that impaired adipose function promotes systemic insulin resistance. The first specific aim examined the hypothesis that activation of AhR suppresses adipogenesis by lowering the rate of pre-adipocyte differentiation. Adipogenesis is a process by which mesenchymal stem cells (MSCs) and pre-adipocytes differentiate into mature adipocytes. Limitations in adipogenesis and accumulation of ectopic lipid have significant roles in decreasing insulin sensitivity. Thus, I hypothesized that POPs contribute to systemic insulin resistance by lowering the rate of MSCs and preadipocyte differentiation; the resulting large, poorly-functioning adipocytes increase serum lipids and promote lipid deposition in other tissues. MSCs derived from mouse bone marrow and pre-adipocytes were treated with different concentrations of AhR agonist, β-Naphthoflavone (BNF), and levels of transcripts associated with adipocyte differentiation were determined by using quantitative PCR. Oil red O staining and lipid content were observed to examine differentiation into mature adipocytes. Genes that promote adipogenesis, including peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer-binding protein alpha (CEBPα), fatty acid binding protein 4 (FABP4), and adiponectin were downregulated in MSCs treated with BNF. Moreover, accumulation of triglycerides was decreased after BNF treatment. Recombinant lentivirus vector-mediated AhR knockdown blocked the effects of BNF on adipogenesis. Therefore, activation of AhR by exogenous ligands inhibits adipogenesis leading to impaired fat storage. Limitations in adipogenesis promotes accumulation of the excess lipid in non-fat tissue such as liver, muscle, and heart leading to decrease the insulin sensitivity and disrupt energy homeostasis. The second specific aim examined effects of AhR activation on circadian clock regulation in adipose tissue. A circadian clock essentially regulates systematic energy homeostasis; the central clock in the suprachiasmatic nucleus (SCN) works with the local clocks in peripheral tissues such as liver, muscle, and adipose tissue to regulate whole-body metabolism. The Tischkau lab has previously shown that AhR interacts with the core machinery of the circadian clock. Activation of AhR by environmental toxicants leads to a dampening of the rhythm expression of core clock genes or an alteration in the timing of their peak expression, which subsequently promotes metabolic disorders such as glucose insensitivity and hyperlipidemia. 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 mature adipose tissue activity. Lipolysis is a clock-regulated process in adipose tissue that provides the necessary energy during periods of fasting and exercise. Thus, I hypothesized that AhR activation in adipose tissue would impair lipolysis by altering molecular circadian clock function. AhR activation was proposed to dampen adipose rhythms, leading to a decreased lipolysis rate during the absence of food, and subsequently, increased glucose concentrations in the blood. C57BL/6 mice were injected with vehicle or 50 mg/kg body weight of the AhR agonist, BNF, 48 hours after release into constant darkness. Mice were sacrificed, and epididymal adipose tissue was collected every 6 hours over a 24 hour period. Real-Time RT-qPCR was used to measure mRNA expression of genes responsible for lipolysis. To examine effects of AhR activation in vitro; mouse pre-adipocytes, 3T3-L1 cells, were differentiated into mature adipocytes for 12 days. Cells were then starved for 24 hours with DMEM media containing 1% FBS to induce lipolysis in the presence of 100, 200, 300 µM of BNF. RNA was then extracted and mRNA expression for genes responsible for circadian clock and lipolysis were determined by RT-qPCR. Alterations were observed in rhythms of core clock genes in wild type mice injected with BNF compared to wild type mice injected with vehicle. Rhythms of key enzymes controlling lipolysis including hormone sensitive lipase (HSL) and adipose triglycerides lipolysis (ATGL) was changed in wild type mice injected with BNF compared to wild type mice injected with vehicle. These effects were blocked in AhR deficient mice, suggesting that these effects were AhR dependent. Liver glycogen was decreased in mice injected with BNF compared to wild type mice injected with vehicle after 12 hour of food restriction but not in AhR null mice. Activation of AhR led to decreased expression of lipolysis genes in adipose tissue at CT6 (middle of the rest phase) as well as in 3T3-L1 cells. Recombinant lentivirus vector-mediated AhR knockdown blocked the effects of BNF on lipolysis in 3T3-L1 cell line. These data establish a link between environmental toxicants and impaired lipolysis, specifically by altering rhythms of clock genes in adipose tissue. In response to the decreased available energy from impaired lipolysis, the body increases glycogenolysis, thereby degrading more glycogen to provide the necessary energy. This process may lead to increased glucose level in the blood and development of type 2 diabetes. The data from this study suggest that activation of AhR by BNF increases the risk of insulin resistance and type 2 diabetes by impairing adipogenesis. Reduced adipogenesis likely decreases adipocyte capacity to capture triglycerides from the blood. These effects may disturb energy homeostasis and contribute to the development of metabolic syndrome. This study also establishes a link between environmental toxicants and impaired lipolysis, specifically by altering rhythms of clock genes in adipose tissue. In response to the decreased available energy from impaired lipolysis, the body increases glycogenolysis, thereby degrading more glycogen to provide the necessary energy. This process may lead to increased glucose level in the blood and development of type 2 diabetes. All together, these data suggest that environmental pollutants result in adipose tissue dysfunction by reducing adipogenesis and lipolysis. Therefore, activation of AHR by its exogenous ligands may increase the risk of insulin resistance and type 2 diabetes by impairing adipose tissue function. In particular, activation of AHR by exogenous ligands leads to impairment of free fatty acids storage during feeding and release during fasting to disturb energy homeostasis.

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