Dysfunctional Nrf2-Keap1 redox signaling in the RVLM is linked to obesity-induced sympathoexcitation.

The rostral ventrolateral medulla (RVLM) is an important region in the brainstem responsible for the tonic and reflex control of sympathetic nerve activity (SNA). Pathological changes at the level of RVLM like oxidative stress has been previously associated with sympathetic nervous system overactivity in obesity. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that provides cytoprotection against oxidative stress by regulating the expression of antioxidant and anti-inflammatory genes. Nrf2 bound to Kelch-like ECH-associated protein 1 (Keap1) in the cytoplasm is inactive and undergoes proteasomal degradation under normal conditions. In response to oxidative stress, Nrf2 dissociates from Keap1, translocates to the nucleus, and forms a complex with the antioxidant response elements (AREs) and small musculoaponeurotic fibrosarcoma protein (smaf) leading to the transcription of multiple antioxidant genes. Previous studies from our lab have shown that increased oxidative stress is associated with Nrf2 dysfunction leading to decreased expression of Nrf2 antioxidant target enzymes in the RVLM of obese mice. The molecular mechanisms of Keap1 in Nrf2 dysfunction and the phenotype of cells undergoing Nrf2 dysfunction at the level of the RVLM in obese animals are unclear. To address these questions, we performed immunofluorescence in 20µm RVLM brainstem sections of C57BL/6J mice fed with a high-fat diet (60kcal% fat) and a chow diet (10kcal% fat) for a period of 16 weeks to investigate the expression of Nrf2 and Keap1. Immunofluorescence showed a higher expression of Keap1 and lower expression of Nrf2 in the RVLM of obese mice when compared with their lean counterparts. To determine the type of cells undergoing Nrf2 dysfunction, an in-vitro cell culture model was developed with human brainstem astrocytes. We hypothesized that brainstem astrocytes are more susceptible to Nrf2 dysfunction mediated oxidative stress. In this experiment, hydrogen peroxide (H O ) was used to mimic diet-induced oxidative stress. Cultured human brainstem astrocytes were treated with H O at the dose rate of 300μM for 2 hrs. Nrf2 and Keap1 levels were assessed by gene expression analysis at the end of 5 days. Data was analyzed by unpaired student's t-test and a p-value less than 0.05 was considered statistically significant. The results showed that H O treatment significantly increased the gene expression levels of Keap1 in human brainstem astrocytes with a trend towards a decrease in the expression of Nrf2. Our results suggest that in obesity, Nrf2-Keap1 dysfunction could occur in astrocytes leading to decreased antioxidant defenses and increased oxidative stress in the RVLM which in turn can cause sympathoexcitation. Future in vivogene transfer studies will investigate the mechanistic role of Nrf2-Keap1 pathway in obesity-induced sympathoexcitation.