NRF as an Oxidative Stress and Nutrient Responsive Transcription Factor in Calorie Restriction

Metabolic syndrome (MetS) can classically be defined by physiological factors, clinical factors, and metabolic factors that are responsible for increased risk of cardiovascular diseases (CVD) and type 2 diabetes (T2D). The International Diabetes Federation has released the most recent definition of MetS, defined by increased waist circumference and two of the following factors: increased serum triglycerides or decreased HDL-cholesterol, increased fasting blood glucose, or hypertension. MetS is also profoundly defined by chronic low-grade inflammation. This proinflammatory state can develop as a result adipocyte hypertrophy and hyperplasia that is responsible for increased secretion of glycerol, free fatty acids, and secretion of adipocytokines. States of adipose tissue dysregulation due to obesity can directly affect systemic inflammation, and the progression to diseased states of insulin resistance and MetS. Adipose tissue does not only contribute to the pathophysiology through proinflammatory signaling, but can alter systemic lipid content by releasing circulating free fatty acids (FFAs) that accumulate in liver, induce insulin resistance in skeletal muscle, and impair pancreatic β-cell functions, all contributing to the potential development of severe metabolic diseases. Most importantly though, oxidative stress has been suggested to be an early event in the pathophysiology and manifestation of chronic metabolic disorders in humans including MetS, T2D, coronary artery disease, and hypertension. The excess storage of lipids in white adipose tissue (WAT), known as obesity, results in oxidative stress and infiltration of macrophages that can alter WAT metabolism. WAT is a metabolically active organ that is essential for proper maintenance of systemic energy balance. WAT is a storage depot for triglycerides, and through hormonal-signaling is responsible for the release of energy to be utilized by tissues, such as, liver and skeletal muscle. In addition to lipid metabolism and mobilization, WAT is responsible for glucose homeostasis, and has major endocrine functions involving secretion of hormones, cytokine, and transcription factors. Due to the major functions of WAT, it is suggested that WAT plays an important role in the development of obesity-related diseases’ requiring greater knowledge and understanding of WAT development, signaling pathways, and its role in systemic diseases. As stated previously, oxidative stress is a unifying characteristic of factors that contribute to MetS, and it is well established that increased lipid content and metabolism can contribute to localized oxidative stress. Metabolic stress within WAT can be defined as the balance between antioxidants and oxidative stress - and publications have begun to demonstrates its effects on adipose tissue, as it relates to metabolic diseases. Another form of oxidative stress is achieved through the generation of reactive oxygen species (ROS) by environmental toxicants. This form of chemical-induced oxidative stress can also be considered important in WAT due to the highly lipophilicity and deposition of persistent organic pollutants in adipose tissue, and the growing concern of these chemicals to act as obesogens. Within this dissertation, Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is addressed as a potential responsive transcription factor to both metabolic stress and nutrient status within a cell, and chemical-induced oxidative stress by persistent lipophilic environmental toxicants in WAT. Nrf2 is a nuclear transcription factor that is known to regulate expression of antioxidant proteins in response to stimulation by oxidative stress or inflammation. Nrf2 is still studied for its cytoprotective effects, but has recently been shown to regulate genes involved in lipid metabolism. Therefore, this overlap in the regulation of antioxidant response and lipid metabolism through antioxidant response element (ARE) activation provides a potential model for the interplay between oxidative stress and metabolic diseases. Our lab has been able to establish the importance of Nf2 in models of steatosis, calorie restriction in the liver, and most recently in obesity and adipogenesis. The purpose of the study performed in MANUSCRIPT I is to elucidate the role of Nrf2 in calorie restriction and identify a potential mechanism of Nrf2-dependent lipid regulation involved in weight loss and decreased fat storage. Few studies exist that have shown Nrf2 activation in adipose tissue (AT), and even fewer studies demonstrate the response of Nrf2 to nutrient status. The data presented in this study demonstrate that 1) The ARE, Nrf2, and classical downstream target expression is induced by calorie restriction in WAT, 2) Nrf2 induction bolsters the antioxidant capacity in vivo and in vitro, 3) Key regulators of calorie restriction and WAT lipid metabolism are induced by an Nrf2-dependent pathway, 4) Important enzymes involved in lipid uptake, lipogenesis, and lipolysis are Nrf2-dependent genes, and 5) CR mimetics and Nrf2 activators can decrease lipid content of adipocytes. In summary, MANUSCRIPT I, demonstrates the importance of increased antioxidant capacity to alleviate factors involved in the development of MetS, such as: glucose intolerance, hypertriglyceridemia, and obesity. The most significant finding is the ability of Nrf2 to respond to the nutrient status of the organism in order to regulate signaling pathways related to energy storage and mobilization. Nrf2 has a novel role in its response it metabolic stressors, nutrient overload, or nutrient deprivation, but the classical activation of Nrf2 by chemical-induced oxidative stress can be reconsidered in understanding how environmental contribute to development of fat mass or obesity. Many environmental toxicants are known to stimulate the ARE/Nrf2 signaling pathway, and it has been considered a hallmark of induction of oxidative stress. Most recently though, environmental chemicals are more persistent in the environment and more lipophilic, therefore being deposited in lipophilic tissues such as AT. The ability of these compounds to activate Nrf2 may not result in an antioxidant response, but can potentially alter the homeostasis of WAT and cause AT dysfunction. AT dysfunction is the hallmark of many diseases, and can be directly to related to obesity. In MANUSCRIPT II, 2,2\u27,4,4\u27,5-pentabromodiphenyl ether (BDE-99) is studied for its ability to induce adipogenesis and lipid development in models of pre-adipocyte differentiation. The ability of the compound to act as an obesogen in WAT, allows for Nrf2 to be studied in a mechanism of chemical-induced regulation of lipid metabolism. Therefore, the purpose of MANUSCRIPT II, was to demonstrate that environmental toxicants can alter Nrf2 expression and subsequent oxidative stress response to alter lipid metabolism in WAT. The observations in this study support 1) The role of BDE-99 in the development of excess lipids during adipocyte differentiation, and 2) A potential mechanism of decreased Nrf2-signaling during differentiation. To summarize, MANUSCRIPT II, demonstrates the ability of an environmental chemical to alter WAT metabolism and contribute to adiposity, through a potential Nrf2-dependent regulation of antioxidant capacity or lipid metabolism directly. Lastly, MANSCURPT III, studies the ability of perinatal deltamethrin exposure to alter WAT metabolism. The focus of this study was to demonstrate that an environmental compound exposure during gestation and alter WAT metabolism and result in phenotypic changes. The observations in the study demonstrate 1) Nrf2 can be induced by chemical-exposure in WAT, 2) Perinatal-exposure to deltamethrin has a hermetic effect on gene expression that does not result in phenotypic changes as adults, 3) Deltamethrin could epigenetically reprogram gene expression, and potentially be involved in the development of susceptibility to obesity or metabolic diseases related to WAT metabolism. In summary, MANUSCRIPT III, represents a perinatal exposure model in which Nrf2 is downregulated in WAT and results in a subsequent down-regulation of genes involved adipogenesis, fat metabolism, and cytokine expression. In conclusion, this dissertation establishes a link between antioxidant response/oxidative stress and WAT metabolism through the ARE/Nrf2 signaling pathway and continues to demonstrate that Nrf2 activation is a hormetic effect and the timing, disease model, and length or intensity of activation need to be considered in order to maintain homeostasis of the vital signaling pathways regulated by Nrf2