We discovered that while Keap1 is directly succinated in the presence of excess fumarate derived from genetic knockdown methods, it is the oxidative modification of Keap1 that predominates in adipocytes matured in high glucose. Kelch-like ECH-associated protein 1 (Keap1) negatively regulates CHOP degradation in the adipocyte, and Keap1 is succinated in cancers where fumarate levels are elevated. We propose that the post-translational modification S-2-succinocysteine may be an alternative physiological regulator of CHOP stability under diabetic conditions. Here we show that CHOP levels are significantly increased in adipocytes matured in high glucose in the absence of UPR signaling and with no sign of apoptosis. Our observations in chapter 2 consistently demonstrated that CHOP levels are elevated in all cases where fumarate and protein succination are increased. The experiments completed in chapter 2 confirm succination of PDI as a novel biochemical mechanism linking altered mitochondrial metabolism to perturbed ER proteostasis in the adipocyte during diabetes. PDI succination and ER stress were decreased, and PDI reductase activity was restored when exposure to chronic high glucose was limited, highlighting the importance of calorie restriction in the improvement of adipocyte metabolic function. Succinated PDI has decreased reductase activity in adipocytes matured in high glucose, and in db/db epididymal adipose tissue, in association with increased levels of the ER stress marker CHOP.
In the presence of prolonged ER stress the unfolded protein response (UPR) triggers the production of the pro-apoptotic protein C/EBP homologous protein (CHOP). PDI is succinated by fumarate on both CXXC containing active sites, contributing to reduced enzymatic activity. The endoplasmic reticulum (ER) oxidoreductase protein disulfide isomerase (PDI) is succinated in adipocytes matured in high glucose, and we investigated if succination would alter PDI oxidoreductase activity, directly linking mitochondrial stress and ER stress. Succination is significantly increased in the adipose tissue of type 2 diabetic mouse models (ob/ob and db/db) and in adipocytes matured in high glucose medium, resulting in impaired protein function. Fumarate can react with cysteine thiol groups to form the chemical modification S-(2-succino)cysteine (2SC), also termed protein succination.
We have shown that this is associated with increased levels of the mitochondrial metabolite fumarate. The intake of excess nutrients surpasses the energy requirements of the cell and leads to increased mitochondrial stress in the adipocyte. Type 2 diabetes mellitus has been diagnosed in ~21 million people in the United States and is closely correlated with obesity, prompting the need for a detailed understanding of adipocyte metabolism in the development of diabetes.
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