Protein Succination‐A Biomarker of Mitochondrial Stress in Adipose Tissue Inhibits Protein Function and is Regulated by Nutrient Balance

Abstract

S‐(2‐succino)cysteine (2SC) is an irreversible post‐translational modification formed by the reaction of fumarate with protein cysteines, also known as protein succination. 2SC is a biomarker of glucotoxicity driven mitochondrial stress in the adipocyte. Here, we investigated if 2SC is a mechanistic mediator of adipocyte stress via modification of the critical chaperone protein disulfide isomerase (PDI) and examined if 2SC levels could be regulated by caloric restriction.Fumarase knockdown or control 3T3‐L1 adipocytes were matured in normal (5 mM) or high (30 mM) glucose for 8 days, or in 30 mM glucose for 4 days and then switched to 5 mM glucose for 4 days. Adipocyte lysates and adipose tissue protein from control and diabetic mice were analyzed by immunoblotting with an anti‐2SC antibody. The fluorescent PDI specific substrate DiEosinGSSG was utilized to assess PDI enzymatic activity in adipocytes. LC‐MS/MS mass spectrometry was utilized to identify the specific site of recombinant PDI succination.2SC significantly increases in adipocytes matured in high glucose and in the adipose tissue of diabetic mice. Succination significantly lowers PDI reductase activity in fumarase knockdown adipocytes and in the adipose tissue of diabetic mice versus control. MS/MS analysis identified an important PDI active site cysteine as a target of succination. 2SC is turned over in adipocytes when maturation medium is returned to normal glucose.2SC inhibits PDI reductase activity facilitating adipocyte stress as PDI is no longer able to re‐fold proteins within the adipocyte. The turnover of succinated proteins when nutrient balance is restored illustrates the importance of a balanced diet in preventing obesity and diabetes‐related dysfunction in adipose tissue. Our data demonstrates a novel biochemical mechanism mediating nutrient induced stress in the adipocyte, and that this stress is ameliorated when reasonable caloric restriction is employed, ensuring healthy functional adipose tissue.Support or Funding InformationNational Institute of Health 1F31DK108559‐01(AMM), 1R56DK105087‐01(NF) and 1‐11‐JF‐13 from the American Diabetes Association (NF).