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Mitochondrial dysfunction and oxidative stress mediate the physiological impairment induced by the disruption of autophagy.

Wu JJ, Quijano C, Chen E, Liu H, Cao L, Fergusson MM, Rovira II, Gutkind S, Daniels MP, Komatsu M, Finkel T - Aging (Albany NY) (2009)

Bottom Line: Using these models we demonstrate that isolated mitochondria obtained from Atg7(-/-) skeletal muscle exhibit a significant defect in mitochondrial respiration.In this model, the simple administration of an antioxidant can significantly ameliorate the physiological impairment in glucose-stimulated insulin secretion.Taken together, these results demonstrate the potential role of mitochondrial dysfunction and oxidative stress in autophagy related pathology.

View Article: PubMed Central - PubMed

Affiliation: Translational Medicine Branch, National Heart Lung and Blood Institute, NIH, Bethesda, MD 20892, USA.

ABSTRACT
Impaired or deficient autophagy is believed to cause or contribute to aging, as well as a number of age-related pathologies. The exact mechanism through which alterations in autophagy induce these various pathologies is not well understood. Here we describe the creation of two in vivo mouse models that allow for the characterization of the alteration in mitochondrial function and the contribution of the corresponding oxidative stress following deletion of Atg7. Using these models we demonstrate that isolated mitochondria obtained from Atg7(-/-) skeletal muscle exhibit a significant defect in mitochondrial respiration. We further show that cells derived from Atg7(-/-) mice have an altered metabolic profile characterized by decreased resting mitochondrial oxygen consumption and a compensatory increase in basal glycolytic rates. Atg7(-/-)cells also exhibit evidence for increased steady state levels of reactive oxygen species. The observed mitochondrial dysfunction and oxidative stress is also evident in a mouse model where Atg7 is deleted within the pancreatic beta cell. In this model, the simple administration of an antioxidant can significantly ameliorate the physiological impairment in glucose-stimulated insulin secretion. Taken together, these results demonstrate the potential role of mitochondrial dysfunction and oxidative stress in autophagy related pathology.

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NAC treatment partially corrects the metabolic defect observed in Atg7 -/- MEFs.                                            (A) Western blot analysis of wild type (+/+) or Atg7-/-                                            MEFs for the expression of Atg7, p62 and actin (loading control) cultured                                            in the presence or absence on the antioxidant NAC (500 μM) for ten days. (B)                                            Primary wild-type and Atg7-/- MEFs that were cultured in the                                            absence or presence of 500 μM NAC for 10                                            days prior to cellular respiration measurement.  Shown is a representative                                            tracing of oxygen consumption performed in triplicate under basal                                            conditions, following the addition of oligomycin (0.5 μM), the                                            pharmacological uncoupler FCCP (1 μM) or the Complex III inhibitor                                            antimycin A (0.25 μM). (C) Averaged metabolic profile from 4                                            separate experiments employing 3 independent primary isolates of WT and                                            Atg7-/- MEFs. Shown is the fold change +/- SEM in oxygen                                            consumption (WT MEF basal respiration =1) for WT MEFs and for Atg7-/-                                            MEFs that were cultured in the absence or presence of 500 μM NAC for 10                                            days prior to metabolic assessment.* p≤0.05; ** p≤0.01; NS= not                                            significant.
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Figure 4: NAC treatment partially corrects the metabolic defect observed in Atg7 -/- MEFs. (A) Western blot analysis of wild type (+/+) or Atg7-/- MEFs for the expression of Atg7, p62 and actin (loading control) cultured in the presence or absence on the antioxidant NAC (500 μM) for ten days. (B) Primary wild-type and Atg7-/- MEFs that were cultured in the absence or presence of 500 μM NAC for 10 days prior to cellular respiration measurement. Shown is a representative tracing of oxygen consumption performed in triplicate under basal conditions, following the addition of oligomycin (0.5 μM), the pharmacological uncoupler FCCP (1 μM) or the Complex III inhibitor antimycin A (0.25 μM). (C) Averaged metabolic profile from 4 separate experiments employing 3 independent primary isolates of WT and Atg7-/- MEFs. Shown is the fold change +/- SEM in oxygen consumption (WT MEF basal respiration =1) for WT MEFs and for Atg7-/- MEFs that were cultured in the absence or presence of 500 μM NAC for 10 days prior to metabolic assessment.* p≤0.05; ** p≤0.01; NS= not significant.

Mentions: Antioxidant treatment did not appear to alter the level of autophagic flux in Atg7-/- MEFs as the level of p62 was unaltered in NAC treated cell (Figure 4A). However, chronic NAC treatment did partially ameliorate the observed metabolic defect seen in these cells (Figure 4 B, C). These results suggest that the continuous oxidative stress observed in Atg7-/- MEFs contributes to the decline in mitochondrial function.


Mitochondrial dysfunction and oxidative stress mediate the physiological impairment induced by the disruption of autophagy.

Wu JJ, Quijano C, Chen E, Liu H, Cao L, Fergusson MM, Rovira II, Gutkind S, Daniels MP, Komatsu M, Finkel T - Aging (Albany NY) (2009)

NAC treatment partially corrects the metabolic defect observed in Atg7 -/- MEFs.                                            (A) Western blot analysis of wild type (+/+) or Atg7-/-                                            MEFs for the expression of Atg7, p62 and actin (loading control) cultured                                            in the presence or absence on the antioxidant NAC (500 μM) for ten days. (B)                                            Primary wild-type and Atg7-/- MEFs that were cultured in the                                            absence or presence of 500 μM NAC for 10                                            days prior to cellular respiration measurement.  Shown is a representative                                            tracing of oxygen consumption performed in triplicate under basal                                            conditions, following the addition of oligomycin (0.5 μM), the                                            pharmacological uncoupler FCCP (1 μM) or the Complex III inhibitor                                            antimycin A (0.25 μM). (C) Averaged metabolic profile from 4                                            separate experiments employing 3 independent primary isolates of WT and                                            Atg7-/- MEFs. Shown is the fold change +/- SEM in oxygen                                            consumption (WT MEF basal respiration =1) for WT MEFs and for Atg7-/-                                            MEFs that were cultured in the absence or presence of 500 μM NAC for 10                                            days prior to metabolic assessment.* p≤0.05; ** p≤0.01; NS= not                                            significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2806022&req=5

Figure 4: NAC treatment partially corrects the metabolic defect observed in Atg7 -/- MEFs. (A) Western blot analysis of wild type (+/+) or Atg7-/- MEFs for the expression of Atg7, p62 and actin (loading control) cultured in the presence or absence on the antioxidant NAC (500 μM) for ten days. (B) Primary wild-type and Atg7-/- MEFs that were cultured in the absence or presence of 500 μM NAC for 10 days prior to cellular respiration measurement. Shown is a representative tracing of oxygen consumption performed in triplicate under basal conditions, following the addition of oligomycin (0.5 μM), the pharmacological uncoupler FCCP (1 μM) or the Complex III inhibitor antimycin A (0.25 μM). (C) Averaged metabolic profile from 4 separate experiments employing 3 independent primary isolates of WT and Atg7-/- MEFs. Shown is the fold change +/- SEM in oxygen consumption (WT MEF basal respiration =1) for WT MEFs and for Atg7-/- MEFs that were cultured in the absence or presence of 500 μM NAC for 10 days prior to metabolic assessment.* p≤0.05; ** p≤0.01; NS= not significant.
Mentions: Antioxidant treatment did not appear to alter the level of autophagic flux in Atg7-/- MEFs as the level of p62 was unaltered in NAC treated cell (Figure 4A). However, chronic NAC treatment did partially ameliorate the observed metabolic defect seen in these cells (Figure 4 B, C). These results suggest that the continuous oxidative stress observed in Atg7-/- MEFs contributes to the decline in mitochondrial function.

Bottom Line: Using these models we demonstrate that isolated mitochondria obtained from Atg7(-/-) skeletal muscle exhibit a significant defect in mitochondrial respiration.In this model, the simple administration of an antioxidant can significantly ameliorate the physiological impairment in glucose-stimulated insulin secretion.Taken together, these results demonstrate the potential role of mitochondrial dysfunction and oxidative stress in autophagy related pathology.

View Article: PubMed Central - PubMed

Affiliation: Translational Medicine Branch, National Heart Lung and Blood Institute, NIH, Bethesda, MD 20892, USA.

ABSTRACT
Impaired or deficient autophagy is believed to cause or contribute to aging, as well as a number of age-related pathologies. The exact mechanism through which alterations in autophagy induce these various pathologies is not well understood. Here we describe the creation of two in vivo mouse models that allow for the characterization of the alteration in mitochondrial function and the contribution of the corresponding oxidative stress following deletion of Atg7. Using these models we demonstrate that isolated mitochondria obtained from Atg7(-/-) skeletal muscle exhibit a significant defect in mitochondrial respiration. We further show that cells derived from Atg7(-/-) mice have an altered metabolic profile characterized by decreased resting mitochondrial oxygen consumption and a compensatory increase in basal glycolytic rates. Atg7(-/-)cells also exhibit evidence for increased steady state levels of reactive oxygen species. The observed mitochondrial dysfunction and oxidative stress is also evident in a mouse model where Atg7 is deleted within the pancreatic beta cell. In this model, the simple administration of an antioxidant can significantly ameliorate the physiological impairment in glucose-stimulated insulin secretion. Taken together, these results demonstrate the potential role of mitochondrial dysfunction and oxidative stress in autophagy related pathology.

Show MeSH
Related in: MedlinePlus