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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. RESEARCH DESIGN AND 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.

OBJECTIVE Reactive oxygen species (ROS) are pro-oxidant factors in distal neurodegeneration in diabetes. We tested the hypothesis that sensory neurons exposed to type 1 diabetes would exhibit enhanced ROS and oxidative stress and determined whether this stress was associated with abnormal axon outgrowth. RESEARCH DESIGN AND METHODS Lumbar dorsal root ganglia sensory neurons from normal or 3- to 5-month streptozotocin (STZ)-diabetic rats were cultured with 10 or 25–50 mmol/l glucose. Cell survival and axon outgrowth were assessed. ROS were analyzed using confocal microscopy. Immunofluorescent staining detected expression of manganese superoxide dismutase (MnSOD) and adducts of 4-hydroxy-2-nonenal (4-HNE), and MitoFluor Green dye detected mitochondria. RESULTS Dorsal root ganglion neurons from normal rats exposed to 25–50 mmol/l glucose did not exhibit oxidative stress or cell death. Cultures from diabetic rats exhibited a twofold (P < 0.001) elevation of ROS in axons after 24 h in 25 mmol/l glucose compared with 10 mmol/l glucose or mannitol. Perikarya exhibited no change in ROS levels. Axonal outgrowth was reduced by approximately twofold (P < 0.001) in diabetic cultures compared with control, as was expression of MnSOD. The antioxidant N-acetyl-cysteine (1 mmol/l) lowered axonal ROS levels, normalized aberrant axonal structure, and prevented deficits in axonal outgrowth in diabetic neurons (P < 0.05). CONCLUSIONS Dorsal root ganglia neurons with a history of diabetes expressed low MnSOD and high ROS in axons. Oxidative stress was initiated by high glucose concentration in neurons with an STZ-induced diabetic phenotype. Induction of ROS was associated with impaired axonal outgrowth and aberrant dystrophic structures that may precede or predispose the axon to degeneration and dissolution in human diabetic neuropathy.

OBJECTIVE—Peripheral neuropathy associated with type 2 diabetes (DPN) is not widely modeled. We describe unique features of DPN in type 2 diabetic Zucker diabetic fatty (ZDF) rats. RESEARCH DESIGN AND METHODS—We evaluated the structural, electrophysiological, behavioral, and molecular features of DPN in ZDF rats and littermates over 4 months of hyperglycemia. The status of insulin signaling transduction molecules that might be interrupted in type 2 diabetes and selected survival-, stress-, and pain-related molecules was emphasized in dorsal root ganglia (DRG) sensory neurons. RESULTS—ZDF rats developed slowing of motor sciatic-tibial and sensory sciatic digital conduction velocity and selective mechanical allodynia with preserved thermal algesia. Diabetic sural axons, preserved in number, developed atrophy, but there was loss of large-calibre dermal and small-calibre epidermal axons. In diabetic rats, insulin signal transduction pathways in lumbar DRGs were preserved or had trends toward upregulation: mRNA levels of insulin receptor β-subunit (IRβ), insulin receptor substrate (IRS)-1, and IRS-2. The numbers of neurons expressing IRβ protein were also preserved. There were trends toward early rises of mRNA levels of heat shock protein 27 (HSP27), the α2δ1 calcium channel subunit, and phosphatidylinositol 3-kinase in diabetes. Others were unchanged, including nuclear factor-κB (NF-κB; p50/p105) and receptor for advanced glycosylation endproducts (RAGE) as was the proportion of neurons expressing HSP27, NF-κB, and RAGE protein. CONCLUSIONS—ZDF type 2 diabetic rats develop a distal degenerative sensory neuropathy accompanied by a selective long-term pain syndrome. Neuronal insulin signal transduction molecules are preserved.