ARRA Supplement P&F Grant Award - Edgar A. Arriaga, Ph.D.

GSTA4 Down-Regulation: A Potential Link Between Mitochondrial Dysfunction and Obesity

Edgar A. Arriaga, Ph.D.
Department of Chemistry, University of Minnesota

Protein carbonylation is increased in adipose tissue in a variety of animal models of obesity and insulin resistant (Grimsrud et al., Mol. Cell Proteomics, 2007). In these models, deficiencies in antioxidant defense systems could contribute to increased protein carbonylation. Evidence indicates that two of the downregulated systems are glutathione S‐transferase (GST) A4 and GSTA3, the enzymes primarily responsible for metabolism of trans‐4‐ hydroxy‐2‐nonenal (4‐HNE). 4‐HNE covalently modifies the side chains of histidine, cysteine and lysine residues of target proteins resulting in their carbonylation that typically leads to loss of protein function and targeted degradation by the 26S proteosome. Paralleling the down regulation of murine GSTA4, human GSTA4 (but not GSTA3) expression is also decreased in adipose tissue from obese insulin resistant subjects. Decreased murine GSTA4 expression is specific to white adipose tissue of C57Bl/6J mice and is targeted to the adipocyte and not stromal‐vascular fraction. When endogenous GSTA4 mRNA is silenced in 3T3‐L1 cells, knockdown adipocytes exhibited increased protein carbonylation, increased lactate production, dysfunctional mitochondrial state 3 respiration and increased levels of matrix superoxide. Extending these findings to animal models, mitochondrial respiration was significantly down regulated in obese (but not lean) C57Bl/6J mice as well as in both lean and obese GSTA4 knockout animals. These results suggest the following hypothesis: down regulation of GSTA4 in murine and human obesity leads to increased protein carbonylation, decreased mitochondrial function, increased oxidative stress and contributes to the development of obesity‐linked type 2 diabetes. To test this hypothesis we propose the following two aims: Aim 1. Identify carbonylated proteins using biochemical and proteomic methods from mitochondria of lean and obese C57Bl/6J mice and compare these to proteins carbonylated in GSTA4 null mice. Aim 2. Assess mitochondrial function and the production of reactive oxygen species in organelles isolated from lean and obese mice.