Glucose-mediated tyrosine nitration in adipocytes: targets and consequences

Abstract

Hyperglycemia, a key factor in insulin resistance and diabetic pathology, is associated with cellular oxidative stress that promotes oxidative protein modifications. We report that protein nitration is responsive to changes in glucose concentrations in 3T3-L1 adipocytes. Alterations in the extent of tyrosine nitration as well as the cellular nitroproteome profile correlated tightly with changing glucose concentrations. The target proteins we identified are involved in fatty acid binding, cell signaling, protein folding, energy metabolism, antioxidant capacity, and membrane permeability. The nitration of adipocyte fatty acid binding protein (FABP4) at Tyr19 decreases, similar to phosphorylation, the binding of palmitic acid to the fatty acid-free protein. This potentially alters intracellular fatty acid transport, nuclear translocation of FABP4, and agonism of PPAR gamma. Our results suggest that protein tyrosine nitration may be a factor in obesity, insulin resistance, and the pathogenesis of diabetes.

Fig. 1

Glucose concentration-dependent increase in protein tyrosine nitration. Representative 2D blots for 3-nitrotyrosine immunoreactivity of 3T3-L1 adipocytes sampled after 24 h of exposure to 5 (A), 6.5 (B), 8 (C), and 11 (D) mM glucose. Cells have been cultured at 5 mM glucose prior to the experiment.

Fig. 2

Increase in protein tyrosine nitration by fluctuating glucose levels. Representative 2D blots for 3-nitrotyrosine immunoreactivity of 3T3-L1 adipocytes sampled after exposure to specified conditions. Cells have been cultured at 5 mM glucose prior to the experiment. Conditions for glucose exposure were: (A) 12 h 5 mM with media change after 2, 4, 6, and 10 h; (B) 2 h 11 mM, 2 h 5 mM, 2 h 11 mM, and 4 h 5 mM; (C) 2 h 11 mM, 2 h 5 mM, 2 h 11 mM, 4 h 5 mM, and 2 h 11 mM.

Fig. 3

Identification of tyrosine 19 as a nitration site in FABP4 using capillary column LC-tandem mass spectrometry. Collision-induced dissociation (CID) spectra of the tryptic peptide peptide 10-LVSSENFDDYMK-21 from FABP4 (NCBI Accession No. 14149635). Representative CID spectra of the unmodified peptide (LVSSENFDDYMK), methionine oxidized peptide (LVSSENFDDYMoK), and tyrosine nitrated-methionine oxidized peptide (LVSSENFDDY^NO2MoK) clearly showing the site of the modified amino acids Tyr19 and Met20. The interpretation of each spectrum is inset.

Fig. 4

Fatty acid binding to FABP4. The binding of [9,10-³H]palmitic acidto nonnitrated and nitrated FABP4 is shown dependent on the applied peroxynitrite concentration. The data are represented as mean±SEM of 4 independent experiments (n=4). Thereby asterisks represent alterations with P<0.02 and were therefore considered significant.