Simultaneous exposure of rats to dioxin and carbon monoxide reduces the xenobiotic but not the hypoxic response.

Aryl hydrocarbon receptor (AhR) and hypoxia-inducible factor-1alpha (HIF-1alpha) are conditionally regulated transcription factor subunits that form heterodimeric complexes with their common partner, AhR nuclear translocator (ARNT/HIF-1beta). Whereas the environmentally toxic compound 2,3,7,8-tetra-chlorodibenzo-p-dioxin (TCDD) initiates the trans-activation activity of AhR:ARNT/HIF-1beta, hypoxic exposure stabilizes HIF-1alpha and functionally activates the HIF-1alpha:ARNT/HIF-1beta complex. To analyze a possible crosstalk between these two pathways in vivo, rats were given dioxin orally and/or were exposed to carbon monoxide (CO), causing functional anemia. We found that exposure to CO inhibited the xenobiotic response while dioxin application had no significant negative impact on hypoxia-mediated gene transcription

Impacts of Oil Exposure During Early Life Development Stages In Sheepshead Minnows (\u3ci\u3eCyprinodon variegatus\u3c/i\u3e) Under Different Environmental Factors

The release of approximately 5 million barrels of crude oil into the northern Gulf of Mexico during the 2010 Deepwater Horizon oil spill jeopardized estuarine ecosystem health from Texas to Florida. These estuarine habitats, which serve as nurseries for many important fisheries are also prone to rapid fluctuations in environmental stressors such as oxygen concentration, and salinity. The consequence of combined exposure to crude oil and suboptimal environmental factors during early life stage development of fish is still largely unknown. The objective of this project was to investigate the impacts of exposure to crude oil in combination with varying environmental stressors on Cyprinodon variegatus survival, gene expression, and genotoxicity. The post-larval developmental stage was the most sensitive early life stage to oil and abiotic stress. Median lethal concentrations during the post-larval exposures followed a treatment dependent pattern with the greatest lethal effect seen under hypoxic-high salinity conditions (64.55 µg/L ± 12.81). Real-time PCR analysis identified down-regulation of cyp1a1, epo, and arnt1, target genes involved in the two common defense pathways, the aryl hydrocarbon receptor signaling pathway which modulates metabolism of polycyclic aromatic hydrocarbons (PAHs), and the hypoxia inducible 1-α signaling pathway which is responsible for resilience to hypoxic stress, this was only observed under hypoxic-high salinity environmental conditions in treatments with PAH concentrations greater than 226 µg/L. Top toxicological functions impacted during post-larval development in all treatment comparisons included cholesterol biosynthesis, cardiotoxicity, and hepatoxicity. These findings indicate that the post-larval stage is the most sensitive to oil and environmental stress

Regulation of the Subunits of Hypoxia Inducible Factors by Sprouty2 and Its Impact on Different Biological Processes

The hypoxia inducible factors (HIF1α, HIF2α, HIF1β) promote transcription of genes that regulate glycolysis and cell survival and growth. Sprouty2 (Spry2) is a modulator of receptor tyrosine kinase signaling and inhibits cell proliferation via different mechanisms. Because of the seemingly opposite actions of the HIF and Spry2 on cellular processes, we hypothesized that Spry2 decreases the protein levels of HIF1α, HIF2α and HIF1β by enhancing the proximity of the HIF subunit to an ubiquitin ligase capable of degrading the subunit. Focusing on HIF1α as a prototypical alpha subunit, in a variety of tumor derived cell lines, Spry2 decreases the protein levels of HIF1α. We showed that this decrease is cause by Spry2 increasing the ubiquitylation and proteosomal degradation of HIF1α by enhancing the amount of pVHL bound to HIF1α. Spry2 also decreases in the mRNA levels of the HIF1α-regulated genes; specifically the genes that regulate glycolysis. Along the same lines, we demonstrated Spry2 decreases the HIF1α-sensitive glucose uptake. In a cell type dependent manner, Spry2 reduced the protein levels of the other half of the transcription factor, HIF1β or Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT), by enhancing proteosomal degradation of ARNT. Spry2 also associates with ARNT. We showed that Nedd4-1, a ubiquitin ligase, regulates ARNT and participates in the ability of Spry2 to regulate the protein levels of ARNT. As a functional impact, we demonstrated that Spry2 reduced the mRNA levels of the ARNT/Aryl hydrocarbon receptor (AhR) regulated gene cytochrome P450 1A1 (CYP1A1), which regulates metabolism of environmental toxins. Together, these data suggest that Spry2 via Nedd4-1 enhances the proteosomal degradation of ARNT decreasing the mRNA levels of ARNT/AhR regulated genes. Overall, my dissertation work has unveiled a novel mode of action for Spry2 by which Spry2 enhances the degradation of proteins that regulate gene transcription by associating with both the target protein and E3 ligase that ubiquitylates the target protein. Also, we have shown a new role for Spry2 in modulating biological processes regulated by HIF subunits, such as glucose uptake. This paradigm could contribute towards the tumor suppressive actions of Spry2 and has major implications in toxicology

Mechanistic Role of ARNT/HIF-1β in the Regulation of Glucose-Stimulated Insulin Secretion

Loss of glucose-stimulated insulin secretion (GSIS) from the pancreatic beta-cells is one of the earliest detectable defects in the pathogenesis of type 2 diabetes. However, despite its relevance, the mechanisms that govern GSIS are still not completely understood. ARNT/HIF-1β is a member of the bHLH-PAS family of transcription factors, with a prominent role in the transcriptional regulation of enzymes required for the metabolism of xenobiotics as well as regulation of genes that are critical for cellular responses to hypoxia. Recent research has uncovered a previously unknown function for ARNT/HIF-1β in the pancreatic beta-cells, where the gene was found to be 90% down-regulated in human type 2 diabetic islets and loss of ARNT/HIF-1β protein leads to defective GSIS in pancreatic beta-cells of mice. The main focus of this thesis was to understand the mechanisms by which ARNT/HIF-1β maintains normal GSIS from pancreatic beta-cells and understand how loss of ARNT/HIF-1β leads to beta-cell dysfunction and type 2 diabetes in mice. ARNT/HIF-1β was found to positively regulate GSIS in both INS-1 derived 832/13 cell line and mice islets. In the 832/13 cells, loss of ARNT/HIF-1β leads to a reduction in glycolysis without affecting the glucose oxidation and the ATP/ADP ratio suggesting that the regulation of GSIS takes place in a manner that is independent of the KATP channels. In order to further assess the mechanism of lowered GSIS in the absence of ARNT/HIF-1β in the 832/13 cells, a metabolite profiling was performed which revealed a significant reduction in the metabolite levels of glycolysis and the TCA cycle intermediates and glucose-induced fatty acid production, suggesting the involvement of ARNT/HIF-1β in regulating glucose-stimulated anaplerosis, which is believed to play a key role in the regulation of GSIS from the pancreatic beta-cells. The changes in metabolite levels in the absence of ARNT/HIF-1β were associated with corresponding changes in the gene expression pattern of key enzymes regulating glycolysis, the TCA cycle and fatty acid synthesis in beta-cells. In an attempt to understand how loss of ARNT/HIF-1β leads to beta-cell dysfunction and type 2 diabetes in mice, a pancreatic beta-cell specific ARNT/HIF-1β knock out mouse (β-ARNT KO) was generated using the Cre-loxP technology. Functional characterization of islets from both male and female β-ARNT KO mice revealed a significant impairment in GSIS, which was attributed due to a small, but significant reduction in rise in intracellular calcium upon glucose stimulation. Further analysis revealed reduced secretory response to glucose in the presence of KCl and diazoxide indicating a defect in the amplifying pathway of GSIS in β-ARNT KO islets. Expression of pyruvate carboxylase (PC) was significantly reduced in β-ARNT KO islets suggesting possible impairments in anaplerosis and consistent with this, defect in GSIS in β-ARNT KO islets could be almost completely rescued by treatment with membrane permeable TCA intermediates. Surprisingly, both male and female β-ARNT KO mice have normal glucose homeostasis. In an attempt to assess how β-ARNT KO mice maintained normal blood glucose levels, indirect calorimetry was used to understand changes in whole-body energy expenditure. This investigation revealed that β-ARNT KO mice exhibited a small but significant increase in respiratory exchange ratio (RER), suggesting a preference in utilizing carbohydrates as a fuel source, possibly leading to improved glucose uptake from the blood stream. Response to exogenous insulin was completely normal in β-ARNT KO mice suggesting intact functioning of the skeletal muscles. To conclude, based on our in vitro data, we believe that ARNT/HIF-1β plays an indispensable role in maintaining normal beta-cell secretory function, however, results from β-ARNT KO mice indicates that these mice are protected from the adverse effects of hyperglycemia. Although loss of ARNT/HIF-1β alone is not sufficient for the genesis of type 2 diabetes, it creates a perfect storm in the pancreatic beta-cells that may eventually lead to an imbalance in the whole body glucose homeostasis. Our study provides significant information to the scientific community that engages in assessing the pharmacological potential of gene targets for the treatment of type 2 diabetes.1 yea