Mitochondrial preparations from DRG were isolated as described ( 8, 21). These data support the conclusion that glucose-dependent superoxide production within the mitochondrial matrix is not a major contributor to oxidative stress in axons of DRG in long-term diabetes. We demonstrate that in diabetes impaired respiratory chain function correlates with decreased protein expression, and in mitochondria of axons, these deficits are associated with membrane depolarization and reduced respiratory chain-derived ROS generation.

To this end, we exploited the use of stable isotope labeling of cells in culture (SILAC) ( 19) to provide a set of culture-derived isotope tags ( 20) to serve as internal standards for a quantitative proteomic analysis. Our previous work on sensory neurons in diabetes demonstrated mitochondrial depolarization within the perikarya and lowered respiratory chain capacity ( 6, 8) however, our objective in the current work was to relate the effects of diabetes on mitochondrial proteome expression to mitochondrial physiology and function within the axon where oxidative stress and degeneration are most clearly defined ( 16, – 18). Since DRG sensory neurons are highly susceptible to glucotoxicity ( 15), we examined the effect of diabetes on the mitochondrial proteome, respiratory capacity, and superoxide production. For example, proteins associated with oxidative phosphorylation were more depressed and respiratory activity decreased in heart compared with liver mitochondria from diabetic Akita mice ( 14). Indeed, the tissue-specific nature of mitochondrial remodeling in diabetes is directly underscored by results from an unbiased proteomic study that identified a differential effect of diabetes on the mitochondrial protein expression and oxidative capacity. Therefore, production of mitochondrial superoxide may exhibit fundamental differences in cells that are targets of diabetic complications. In addition, activities of citrate synthase and mitochondrial respiratory chain are decreased in the skeletal muscle of patients with type 2 diabetes ( 11, – 13). These findings are aligned with studies in diabetic heart where mitochondrial respiration and enzymatic activities are reduced ( 9, 10). Further, lumbar dorsal root ganglia (DRG) from diabetic rats exhibit reduced respiratory chain activity that correlate with the downregulation of select proteins within the electron transport chain complexes ( 8). Similarly, in sensory neurons from STZ-diabetic rats, the mitochondrial membrane potential is depolarized and not hyperpolarized, as observed in endothelial cells exposed to hyperglycemia ( 6, 7). On the other hand, studies in diabetic retina suggest that metabolism of high glucose concentrations does not operate in a fashion that supports superoxide formation by the respiratory chain ( 5).

In support of this mechanism, epineurial arterioles serving the sciatic nerve of STZ-diabetic rats show increased levels of mitochondrial superoxide that is dependent on complex I activity ( 4). These investigators proposed that hyperglycemia increases mitochondrial NADH levels and that increased electron availability and/or saturation causes partial reduction of oxygen to superoxide in the proximal part of the respiratory chain ( 2). In cultured endothelial cells, hyperglycemia induced excessive electron flux through the respiratory chain that promoted mitochondrial hyperpolarization and elevated ROS production ( 2, 3). ( 2) suggested that mitochondrial superoxide generation may be a critical feature in the onset of diabetic complications. To develop more targeted therapeutics toward ameliorating oxidative stress and the development of diabetic peripheral neuropathy, considerable effort has focused on identifying the cellular source of reactive oxygen species (ROS) over the past decade. Enhanced oxidative stress is thought to be a central pathologic feature in the etiology of diabetic peripheral neuropathy ( 1).

Superoxide d splitt