Mitochondrial Respiratory Chain Dysfunction in Dorsal Root Ganglia of Streptozotocin-Induced Diabetic Rats and Its Correction by Insulin Treatment

OBJECTIVE

Impairments in mitochondrial physiology may play a role in diabetic sensory neuropathy. We tested the hypothesis that mitochondrial dysfunction in sensory neurons is due to abnormal mitochondrial respiratory function.

RESEARCH DESIGN AND METHODS

Rates of oxygen consumption were measured in mitochondria from dorsal root ganglia (DRG) of 12- to- 22-week streptozotocin (STZ)-induced diabetic rats, diabetic rats treated with insulin, and age-matched controls. Activities and expression of components of mitochondrial complexes and reactive oxygen species (ROS) were analyzed.

RESULTS

Rates of coupled respiration with pyruvate + malate (P + M) and with ascorbate + TMPD (Asc + TMPD) in DRG were unchanged after 12 weeks of diabetes. By 22 weeks of diabetes, respiration with P + M was significantly decreased by 31–44% and with Asc + TMPD by 29–39% compared with control. Attenuated mitochondrial respiratory activity of STZ-diabetic rats was significantly improved by insulin that did not correct other indices of diabetes. Activities of mitochondrial complexes I and IV and the Krebs cycle enzyme, citrate synthase, were decreased in mitochondria from DRG of 22-week STZ-diabetic rats compared with control. ROS levels in perikarya of DRG neurons were not altered by diabetes, but ROS generation from mitochondria treated with antimycin A was diminished compared with control. Reduced mitochondrial respiratory function was associated with downregulation of expression of mitochondrial proteins.

CONCLUSIONS

Mitochondrial dysfunction in sensory neurons from type 1 diabetic rats is associated with impaired rates of respiratory activity and occurs without a significant rise in perikaryal ROS.Peripheral nerves of diabetic patients develop a spectrum of pathology that can take many years to emerge (1,2). Experimental studies over shorter time periods have demonstrated that hyperglycemia can induce cellular damage through increased glucose metabolism by aldose reductase (3), elevated protein glycation (4), and increased mitochondrial NADH supply, which enhances electron availability causing partial reduction of oxygen to superoxide radicals in the proximal part of the electron transport chain (5,6). These three mechanisms may combine to trigger large elevations in reactive oxygen species (ROS) that induce oxidative stress and tissue damage (7–9). The ability of nerves to survive oxidative stress may also be impaired in diabetes because of suboptimal trophic support from insulin, insulin-like growth factors, and neurotrophic factors (10,11).Although increased oxidative stress has become a widely accepted consequence of hyperglycemia in models of diabetic neuropathy, the source of excessive ROS is not defined. Mitochondria are one plausible source of ROS, based on reports of an acute glucose-induced elevation of flux through the electron transport chain in endothelial cells that was accompanied by hyperpolarization of the mitochondrial inner membrane and subsequent stimulation of ROS generation in the proximal aspect of the electron transport chain (5,6). However, this mechanism may not extend to other cell types or to longer durations of diabetes, since mitochondrial respiration and enzymatic activities are reduced in mitochondria from streptozotocin (STZ)-induced diabetic rat hearts (12,13), and activity of the mitochondrial electron transport chain and citrate synthase are decreased in skeletal muscle of patients with type 2 diabetes (14–16). Further, we and others have measured depolarization, rather than hyperpolarization, of the mitochondrial inner membrane in acutely isolated adult DRG sensory neurons from STZ-diabetic rats that can be corrected by treatment with neurotrophin-3 or insulin at doses that did not affect hyperglycemia (17–19). Given the unclear understanding of the contribution of mitochondrial-derived ROS to neuronal oxidative stress, we have now directly measured rates of activity within the mitochondrial respiratory chain and ROS production in neurons from STZ-diabetic rats.