217 - Protein Succination Exacerbates Oxidative Stress and Mediates CHOP Stability in the Adipocyte during Diabetes

Abstract

The intake of excess nutrients surpasses the energy requirements of the adipocyte and leads to increased mitochondrial stress. We have previously shown that this is associated with the accumulation of the mitochondrial metabolite fumarate. Fumarate can react with protein thiols to form the chemical modification S-(2-succino) cysteine (2SC), also termed protein succination. Succination is significantly increased in the adipose tissue of type 2 diabetic mouse models, and in adipocytes matured in high glucose, resulting in impaired protein function. We consistently observe that the transcription factor CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) is elevated in parallel with increased fumarate and protein succination, independent of other markers of endoplasmic reticulum stress. We propose that fumarate may indirectly promote CHOP stability through the succination of Keap1 thiols, as Keap1 regulates CHOP ubiquitination and turnover. Fumarase knockdown (FHKD) or control 3T3-L1 adipocytes were matured in normal (5 mM) or high (25-30 mM) glucose. Protein succination, CHOP stability and activation of Keap1/Nrf2 in adipocyte lysates and adipose tissue (db/db mice) were analyzed. Keap1 succination sites were identified by LC-MS/MS. Fumarate, glutathione, and reactive oxygen species were measured in control and FHKD adipocytes. We discovered that while Keap1 is directly succinated in the presence of excess fumarate derived from fumarase knockdown cells, it is the oxidative modification of Keap1 that predominates in adipocytes matured in high glucose. We also determined that succination indirectly regulates CHOP stability through the induction of oxidative stress in the FHKD model. N-acetylcysteine reduced fumarate levels, protein succination and CHOP levels in adipocytes matured in high glucose. The results shown here demonstrate that elevated fumarate can induce oxidative stress, and that the oxidation of Keap1 thiols contributes to reduced CHOP turnover in the adipocyte during diabetes. This study emphasizes the combined roles of both metabolite derived stress and oxidative stress in the modulation of adipocyte proteins during diabetes.