Carbonylation of adipose proteins in obesity and insulin resistance: identification of adipocyte fatty acid-binding protein as a cellular target of 4-hydroxynonenal.
Obesity is a state of mild inflammation correlated with increased oxidative stress. In general, pro-oxidative conditions lead to production of reactive aldehydes such as trans-4-hydroxy-2-nonenal (4-HNE) and trans-4-oxo-2-nonenal implicated in the development of a variety of metabolic diseases. To investigate protein modification by 4-HNE as a consequence of obesity and its potential relationship to the development of insulin resistance, proteomics technologies were utilized to identify aldehyde-modified proteins in adipose tissue. Adipose proteins from lean insulin-sensitive and obese insulin-resistant C57Bl/6J mice were incubated with biotin hydrazide and detected using horseradish peroxidase-conjugated streptavidin. High carbohydrate, high fat feeding of mice resulted in a approximately 2-3-fold increase in total adipose protein carbonylation. Consistent with an increase in oxidative stress in obesity, the abundance of glutathione S-transferase A4 (GSTA4), a key enzyme responsible for metabolizing 4-HNE, was decreased approximately 3-4-fold in adipose tissue of obese mice. To identify specific carbonylated proteins, biotin hydrazide-modified adipose proteins from obese mice were captured using avidin-Sepharose affinity chromatography, proteolytically digested, and subjected to LC-ESI MS/MS. Interestingly enzymes involved in cellular stress response, lipotoxicity, and insulin signaling such as glutathione S-transferase M1, peroxiredoxin 1, glutathione peroxidase 1, eukaryotic elongation factor 1alpha-1 (eEF1alpha1), and filamin A were identified. The adipocyte fatty acid-binding protein, a protein implicated in the regulation of insulin resistance, was found to be carbonylated in vivo with 4-HNE. In vitro modification of adipocyte fatty acid-binding protein with 4-HNE was mapped to Cys-117, occurred equivalently using either the R or S enantiomer of 4-HNE, and reduced the affinity of the protein for fatty acids approximately 10-fold. These results indicate that obesity is accompanied by an increase in the carbonylation of a number of adipose-regulatory proteins that may serve as a mechanistic link between increased oxidative stress and the development of insulin resistance.
Grimsrud
Paul A
PA
Department of Biochemistry, Molecular Biology and Biophysics, The University of Minnesota, Minneapolis, Minnesota 55455, USA.
Picklo
Matthew J
MJ
Griffin
Timothy J
TJ
Bernlohr
David A
DA
D000273:Adipose Tissue / Q000378:metabolism*; D000447:Aldehydes / Q000378:metabolism*; D000818:Animals; D050556:Fatty Acid-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D007333:Insulin Resistance / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D009765:Obesity / Q000378:metabolism*; D050050:Protein Carbonylation; D040901:Proteomics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry
D000447:Aldehydes; D050556:Fatty Acid-Binding Proteins; D005982:Glutathione Transferase; C041116:leukotriene-C4 synthase; C027576:4-hydroxy-2-nonenal