Investigating the role of transcription factors Nrf2 and Pparα in hepatic lipid metabolism during fasting
The transcription factor nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) controls the basal and inducible expression of a plethora of genes, predominantly those associated with oxidative stress response through its regulation of the glutathione and thioredoxin based antioxidant systems, and the maintenance of cellular redox homeostasis. While Nrf2 has long been studied in this context, it has more recently become apparent that the role of Nrf2 goes far beyond this narrow spectrum, including a clear role in the regulation of pathways involved in NADPH generation, gluconeogenesis, and lipid metabolism, although the true extent of the contribution of Nrf2 in vivo to many of these processes has yet to be fully elucidated. When the Nrf2 gene is disrupted, many genes involved in lipogenesis, which are known to be regulated by Pparα, are upregulated, indicating a potential role for Nrf2 in the repression of Pparα, a master regulator of hepatic lipid metabolism. The primary aim of this thesis was to further define the role of Nrf2 during metabolic stress and to determine the mechanism of action of such a function. Genetically altered animals of the genotypes WT, Nrf2-/-, Pparα-/- and Nrf2-/-/::Pparα-/- double knockout were utilised during the course of this study in an effort to tease apart the interactions of transcription factors Nrf2 and Pparα under acute and extended fasting conditions, when Pparα is strongly activated, to better understand their in vivo relationship. Data presented within this thesis demonstrate that during periods of extended fasting, Nrf2 serves to diminish the magnitude of gene induction mediated by Pparα in response to fasting. In Nrf2-/- animals during 48 hr fasting, the level of Pparα target gene expression is significantly higher, with corresponding lower plasma free fatty acid concentration than their WT counterparts. Upon genetic knockout of Pparα the target genes which were upregulated in the Nrf2-/- animals were not induced in response to fasting, indicating that their increased expression in response to fasting is solely regulated by Pparα. In order to further verify that the induction of these fasting-associated genes in the absence of Nrf2 was Pparα-dependent, animals null for both transcription factors were subjected to an extended fast and were shown not to support increased expression of the Pparα target genes despite the absence of Nrf2. Together, these data substantiate our hypothesis that under prolonged fasting conditions, the Pparα response is negatively regulated by Nrf2. Examination of gene induction at the more acute 24 hr time point revealed that Nrf2-/- animals showed similar expression levels of Pparα target genes as WT animals, in addition to exhibiting similar plasma free fatty acid concentrations as their WT counterparts, suggesting that the negative regulation of Pparα by Nrf2 reflects chronic biochemical changes. Additionally, it was demonstrated for the first time that robust activation of Nrf2 occurs in response to prolonged fasting, with highly significant induction of the prototypic Nrf2 target gene Nqo1. Importantly, this activation also occurred in the absence of Pparα, indicating that it is not via direct crosstalk between the transcription factors, nor is it a consequence of the generation of reactive oxygen species by the oxidation of fatty acids. In conclusion, this thesis describes data that provide further insight into the physiological role of Nrf2 in fatty acid metabolism. Future work is required to build upon this foundation and fully elucidate the signal(s) which leads to Nrf2 activation in response to prolonged periods of fasting.
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