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Diminished superoxide generation is associated with respiratory chain dysfunction and changes in the mitochondrial proteome of sensory neurons from diabetic rats.

Akude E, Zherebitskaya E, Chowdhury SK, Smith DR, Dobrowsky RT, Fernyhough P - Diabetes (2010)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: St. Boniface Hospital Research Centre, Winnipeg, Manitoba, Canada.

ABSTRACT

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.

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The mitochondria of DRG sensory neurons exhibited lower respiratory chain activity. A: Oxygen consumption was assessed in freshly isolated mitochondria from lumbar DRG of age-matched control and 22-week-old diabetic rats using an OROBOROS oxygraph 2k. Coupled respiration rates were measured in the presence of pyruvate (P) (10 mmol/l), malate (M) (5.0 mmol/l), and ADP (2.0 mmol/l). The addition of FCCP (0.5 μmol/l) permits a measure of uncoupled respiratory chain activity. Addition of ascorbate (Asc) (5.0 mmol/l) and TMPD (0.5 mmol/l) permit an analysis of complex IV activity that was verified by specific inhibitors. Values are mean ± SEM; n = 5. *P < 0.05 vs. controls; **P < 0.001 vs. controls. B: Images of fluorescence confocal microscopy using TMRM in live cultures of DRG neurons isolated from control adult rats showing effect of antimycin A and oligomycin. C: Trace of TMRM fluorescence signal in the axons of cultured DRG neurons isolated from age-matched controls and STZ-diabetic rats. D: Shows the area under the TMRM fluorescence trace (area under the curve) for control (open bar) and diabetic (filled bar) neurons. The area under the curve was estimated from the baseline (at the point of injection) to a fluorescence level of 0.2 and between time points 1.0 min and 6 min using sums of squares (shown by dotted line). Values are the means ± SEM, n = 65–80 axons; *P < 0.001 compared with control, t test. The TMRM trace was characterized by nonlinear regression (one phase exponential decay). The rate constant of decay (K) = 0.013 ± 0.0004 (control) and 0.006 ± 0.0001 (diabetic). Half-life of decay = 54.19 s (control) and 108.7 s (diabetic). The Fisher parametric (F) ratio = 409.5, P < 0.0001, control vs. diabetic. The F ratio compares the goodness-of-fit of the two curves. The red arrow indicates point of injection of antimycin A + oligomycin. (A high-quality digital representation of this figure is available in the online issue.)
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Figure 3: The mitochondria of DRG sensory neurons exhibited lower respiratory chain activity. A: Oxygen consumption was assessed in freshly isolated mitochondria from lumbar DRG of age-matched control and 22-week-old diabetic rats using an OROBOROS oxygraph 2k. Coupled respiration rates were measured in the presence of pyruvate (P) (10 mmol/l), malate (M) (5.0 mmol/l), and ADP (2.0 mmol/l). The addition of FCCP (0.5 μmol/l) permits a measure of uncoupled respiratory chain activity. Addition of ascorbate (Asc) (5.0 mmol/l) and TMPD (0.5 mmol/l) permit an analysis of complex IV activity that was verified by specific inhibitors. Values are mean ± SEM; n = 5. *P < 0.05 vs. controls; **P < 0.001 vs. controls. B: Images of fluorescence confocal microscopy using TMRM in live cultures of DRG neurons isolated from control adult rats showing effect of antimycin A and oligomycin. C: Trace of TMRM fluorescence signal in the axons of cultured DRG neurons isolated from age-matched controls and STZ-diabetic rats. D: Shows the area under the TMRM fluorescence trace (area under the curve) for control (open bar) and diabetic (filled bar) neurons. The area under the curve was estimated from the baseline (at the point of injection) to a fluorescence level of 0.2 and between time points 1.0 min and 6 min using sums of squares (shown by dotted line). Values are the means ± SEM, n = 65–80 axons; *P < 0.001 compared with control, t test. The TMRM trace was characterized by nonlinear regression (one phase exponential decay). The rate constant of decay (K) = 0.013 ± 0.0004 (control) and 0.006 ± 0.0001 (diabetic). Half-life of decay = 54.19 s (control) and 108.7 s (diabetic). The Fisher parametric (F) ratio = 409.5, P < 0.0001, control vs. diabetic. The F ratio compares the goodness-of-fit of the two curves. The red arrow indicates point of injection of antimycin A + oligomycin. (A high-quality digital representation of this figure is available in the online issue.)

Mentions: Lumbar DRG from age-matched control and 22-week-old STZ-diabetic rats were analyzed for rates of oxygen consumption as shown in Fig. 3A. Respiratory chain activity, whether coupled or uncoupled, was significantly depressed in diabetic samples. In agreement with the proteomic data and oxygen consumption results, the enzymatic activities of rotenone-sensitive NADH-cytochrome c reductase (complex I) and cytochrome c oxidase (COX; complex IV), as well as the Krebs cycle enzyme, citrate synthase, were significantly decreased in STZ-diabetic rats compared with control (supplementary Table 2).


Diminished superoxide generation is associated with respiratory chain dysfunction and changes in the mitochondrial proteome of sensory neurons from diabetic rats.

Akude E, Zherebitskaya E, Chowdhury SK, Smith DR, Dobrowsky RT, Fernyhough P - Diabetes (2010)

The mitochondria of DRG sensory neurons exhibited lower respiratory chain activity. A: Oxygen consumption was assessed in freshly isolated mitochondria from lumbar DRG of age-matched control and 22-week-old diabetic rats using an OROBOROS oxygraph 2k. Coupled respiration rates were measured in the presence of pyruvate (P) (10 mmol/l), malate (M) (5.0 mmol/l), and ADP (2.0 mmol/l). The addition of FCCP (0.5 μmol/l) permits a measure of uncoupled respiratory chain activity. Addition of ascorbate (Asc) (5.0 mmol/l) and TMPD (0.5 mmol/l) permit an analysis of complex IV activity that was verified by specific inhibitors. Values are mean ± SEM; n = 5. *P < 0.05 vs. controls; **P < 0.001 vs. controls. B: Images of fluorescence confocal microscopy using TMRM in live cultures of DRG neurons isolated from control adult rats showing effect of antimycin A and oligomycin. C: Trace of TMRM fluorescence signal in the axons of cultured DRG neurons isolated from age-matched controls and STZ-diabetic rats. D: Shows the area under the TMRM fluorescence trace (area under the curve) for control (open bar) and diabetic (filled bar) neurons. The area under the curve was estimated from the baseline (at the point of injection) to a fluorescence level of 0.2 and between time points 1.0 min and 6 min using sums of squares (shown by dotted line). Values are the means ± SEM, n = 65–80 axons; *P < 0.001 compared with control, t test. The TMRM trace was characterized by nonlinear regression (one phase exponential decay). The rate constant of decay (K) = 0.013 ± 0.0004 (control) and 0.006 ± 0.0001 (diabetic). Half-life of decay = 54.19 s (control) and 108.7 s (diabetic). The Fisher parametric (F) ratio = 409.5, P < 0.0001, control vs. diabetic. The F ratio compares the goodness-of-fit of the two curves. The red arrow indicates point of injection of antimycin A + oligomycin. (A high-quality digital representation of this figure is available in the online issue.)
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Related In: Results  -  Collection

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Figure 3: The mitochondria of DRG sensory neurons exhibited lower respiratory chain activity. A: Oxygen consumption was assessed in freshly isolated mitochondria from lumbar DRG of age-matched control and 22-week-old diabetic rats using an OROBOROS oxygraph 2k. Coupled respiration rates were measured in the presence of pyruvate (P) (10 mmol/l), malate (M) (5.0 mmol/l), and ADP (2.0 mmol/l). The addition of FCCP (0.5 μmol/l) permits a measure of uncoupled respiratory chain activity. Addition of ascorbate (Asc) (5.0 mmol/l) and TMPD (0.5 mmol/l) permit an analysis of complex IV activity that was verified by specific inhibitors. Values are mean ± SEM; n = 5. *P < 0.05 vs. controls; **P < 0.001 vs. controls. B: Images of fluorescence confocal microscopy using TMRM in live cultures of DRG neurons isolated from control adult rats showing effect of antimycin A and oligomycin. C: Trace of TMRM fluorescence signal in the axons of cultured DRG neurons isolated from age-matched controls and STZ-diabetic rats. D: Shows the area under the TMRM fluorescence trace (area under the curve) for control (open bar) and diabetic (filled bar) neurons. The area under the curve was estimated from the baseline (at the point of injection) to a fluorescence level of 0.2 and between time points 1.0 min and 6 min using sums of squares (shown by dotted line). Values are the means ± SEM, n = 65–80 axons; *P < 0.001 compared with control, t test. The TMRM trace was characterized by nonlinear regression (one phase exponential decay). The rate constant of decay (K) = 0.013 ± 0.0004 (control) and 0.006 ± 0.0001 (diabetic). Half-life of decay = 54.19 s (control) and 108.7 s (diabetic). The Fisher parametric (F) ratio = 409.5, P < 0.0001, control vs. diabetic. The F ratio compares the goodness-of-fit of the two curves. The red arrow indicates point of injection of antimycin A + oligomycin. (A high-quality digital representation of this figure is available in the online issue.)
Mentions: Lumbar DRG from age-matched control and 22-week-old STZ-diabetic rats were analyzed for rates of oxygen consumption as shown in Fig. 3A. Respiratory chain activity, whether coupled or uncoupled, was significantly depressed in diabetic samples. In agreement with the proteomic data and oxygen consumption results, the enzymatic activities of rotenone-sensitive NADH-cytochrome c reductase (complex I) and cytochrome c oxidase (COX; complex IV), as well as the Krebs cycle enzyme, citrate synthase, were significantly decreased in STZ-diabetic rats compared with control (supplementary Table 2).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: St. Boniface Hospital Research Centre, Winnipeg, Manitoba, Canada.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus