Diminished superoxide generation is associated with respiratory chain dysfunction and changes in the mitochondrial proteome of sensory neurons from diabetic rats.
OBJECTIVE
Impairments in mitochondrial function have been proposed to play a role in the etiology of diabetic sensory neuropathy. We tested the hypothesis that mitochondrial dysfunction in axons of sensory neurons in type 1 diabetes is due to abnormal activity of the respiratory chain and an altered mitochondrial proteome.


METHODS
Proteomic analysis using stable isotope labeling with amino acids in cell culture (SILAC) determined expression of proteins in mitochondria from dorsal root ganglia (DRG) of control, 22-week-old streptozotocin (STZ)-diabetic rats, and diabetic rats treated with insulin. Rates of oxygen consumption and complex activities in mitochondria from DRG were measured. Fluorescence imaging of axons of cultured sensory neurons determined the effect of diabetes on mitochondrial polarization status, oxidative stress, and mitochondrial matrix-specific reactive oxygen species (ROS).


RESULTS
Proteins associated with mitochondrial dysfunction, oxidative phosphorylation, ubiquinone biosynthesis, and the citric acid cycle were downregulated in diabetic samples. For example, cytochrome c oxidase subunit IV (COX IV; a complex IV protein) and NADH dehydrogenase Fe-S protein 3 (NDUFS3; a complex I protein) were reduced by 29 and 36% (P < 0.05), respectively, in diabetes and confirmed previous Western blot studies. Respiration and mitochondrial complex activity was significantly decreased by 15 to 32% compared with control. The axons of diabetic neurons exhibited oxidative stress and depolarized mitochondria, an aberrant adaption to oligomycin-induced mitochondrial membrane hyperpolarization, but reduced levels of intramitochondrial superoxide compared with control.


CONCLUSIONS
Abnormal mitochondrial function correlated with a downregulation of mitochondrial proteins, with components of the respiratory chain targeted in lumbar DRG in diabetes. The reduced activity of the respiratory chain was associated with diminished superoxide generation within the mitochondrial matrix and did not contribute to oxidative stress in axons of diabetic neurons. Alternative pathways involving polyol pathway activity appear to contribute to raised ROS in axons of diabetic neurons under high glucose concentration.
Akude
Eli
E
St. Boniface Hospital Research Centre, Winnipeg, Manitoba, Canada.
Zherebitskaya
Elena
E
Chowdhury
Subir K Roy
SK
Smith
Darrell R
DR
Dobrowsky
Rick T
RT
Fernyhough
Paul
P
D000818:Animals; D001786:Blood Glucose / Q000378:metabolism; D001835:Body Weight; D002478:Cells, Cultured; D003921:Diabetes Mellitus, Experimental / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism; D004343:Drug Implants; D006442:Glycated Hemoglobin / Q000378:metabolism; D007004:Hypoglycemic Agents / Q000627:therapeutic use; D007328:Insulin / Q000008:administration & dosage / Q000627:therapeutic use; D008297:Male; D008697:Methane / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology*; D051336:Mitochondrial Membranes / Q000502:physiology; D009474:Neurons / Q000166:cytology / Q000502:physiology; D010085:Oxidative Phosphorylation; D010101:Oxygen Consumption; D020543:Proteome / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism; D011984:Sensory Receptor Cells / Q000187:drug effects / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism*; D014451:Ubiquinone / Q000378:metabolism
D001786:Blood Glucose; D004343:Drug Implants; D006442:Glycated Hemoglobin; D007004:Hypoglycemic Agents; D007328:Insulin; D008697:Methane; D020543:Proteome; D017382:Reactive Oxygen Species; D013481:Superoxides; D014451:Ubiquinone