Abstract
C/EBP homologous protein (CHOP) is a transcription factor that is elevated in adipose tissue across many models of diabetes and metabolic stress. Although increased CHOP levels are associated with the terminal response to endoplasmic reticulum stress and apoptosis, there is no evidence for CHOP mediated apoptosis in the adipose tissue during diabetes. CHOP protein levels increase in parallel with protein succination, a fumarate derived cysteine modification, in the adipocyte during metabolic stress. We investigated the factors contributing to sustained CHOP proteins levels in the adipocyte, with an emphasis on the regulation of CHOP protein turnover by metabolite-driven modification of Keap1 cysteines. CHOP protein stability was investigated in conditions of nutrient stress due to high glucose or elevated fumarate (fumarase knockdown model); where cysteine succination is specifically elevated. CHOP protein turnover is significantly reduced in models of elevated glucose and fumarate with a ~30% increase in CHOP stability (p > 0.01), in part due to decreased CHOP phosphorylation. Sustained CHOP levels occur in parallel with elevated heme-oxygenase-1, a production of increased Nrf2 transcriptional activity and Keap1 modification. While Keap1 is directly succinated in the presence of excess fumarate derived from genetic knockdown of fumarase (fumarate levels are elevated >20-fold), it is the oxidative modification of Keap1 that predominates in adipocytes matured in high glucose (fumarate increases 4-5 fold). Elevated fumarate indirectly regulates CHOP stability through the induction of oxidative stress. The antioxidant N-acetylcysteine (NAC) reduces fumarate levels, protein succination and CHOP levels in adipocytes matured in high glucose. Elevated CHOP does not contribute elevated apoptosis in adipocytes, but plays a redox-dependent role in decreasing the adipocyte secretion of interleukin-13, an anti-inflammatory chemokine. NAC treatment restores adipocyte IL-13 secretion, confirming the redox-dependent regulation of a potent anti-inflammatory eotaxin. This study demonstrates that physiological increases in the metabolite fumarate during high glucose exposure contributes to the presence of oxidative stress and sustained CHOP levels in the adipocyte during diabetes. The results reveal a novel metabolic link between mitochondrial metabolic stress and reduced anti-inflammatory adipocyte signaling as a consequence of reduced CHOP protein turnover.