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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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Alterations in the energetics of Atg7 -/- MEFs.                                            (A) Western blot                                            analysis of wild type (+/+) or Atg7-/- MEFs for the expression                                            of Atg7, p62 and actin (loading control). (B) Measurement of oxygen                                            consumption for WT and Atg7-/-  MEFs under basal conditions,                                            following the addition of the mitochondrial electron chain inhibitor                                            oligomycin (0.5 μM), or in the presence of the mitochondrial uncoupler FCCP                                            (1 μM), to determine maximal oxidative capacity.  Shown is the average fold                                            change +/- SEM in oxygen consumption (WT MEFs basal respiration=1) obtained                                            from 5 experiments each performed in triplicate. (C)  Assessment of                                            mitochondrial number in WT or Atg7-/- MEFs. DNA was isolated                                            from WT (n=3 independent WT MEF cell isolates) and Atg7-/- MEFs (n=3                                            independent Atg7-/- MEF cell isolates) and quantitative PCR                                            analysis performed for the mitochondrial-encoded gene ND1 and the                                            nuclear-encoded gene H19.  (D) Relative extracellular acidification                                            rates indicating lactic acid production and hence glyolytic rates in WT or                                            Atg7-/- MEFs.  Shown is the average +/- SEM fold change in                                            lactic acid production from 8 experiments each performed in triplicate. *                                            p≤0.05; ** p≤0.01.
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Figure 2: Alterations in the energetics of Atg7 -/- MEFs. (A) Western blot analysis of wild type (+/+) or Atg7-/- MEFs for the expression of Atg7, p62 and actin (loading control). (B) Measurement of oxygen consumption for WT and Atg7-/- MEFs under basal conditions, following the addition of the mitochondrial electron chain inhibitor oligomycin (0.5 μM), or in the presence of the mitochondrial uncoupler FCCP (1 μM), to determine maximal oxidative capacity. Shown is the average fold change +/- SEM in oxygen consumption (WT MEFs basal respiration=1) obtained from 5 experiments each performed in triplicate. (C) Assessment of mitochondrial number in WT or Atg7-/- MEFs. DNA was isolated from WT (n=3 independent WT MEF cell isolates) and Atg7-/- MEFs (n=3 independent Atg7-/- MEF cell isolates) and quantitative PCR analysis performed for the mitochondrial-encoded gene ND1 and the nuclear-encoded gene H19. (D) Relative extracellular acidification rates indicating lactic acid production and hence glyolytic rates in WT or Atg7-/- MEFs. Shown is the average +/- SEM fold change in lactic acid production from 8 experiments each performed in triplicate. * p≤0.05; ** p≤0.01.

Mentions: In order to further characterize the defect in mitochondrial function within the context of intact cells, we next isolated mouse embryonic fibroblasts (MEFs) from wild type or Atg7-/- embryos (Figure 2A). Compared to WT MEFs, Atg7-/- MEFs exhibited a reduction in basal oxygen consumption (Figure 2B). In addition, we noted that Atg7-/- MEFs demonstrated a marked reduction in maximal mitochondrial oxidative capacity, as assessed by the levels of FCCP-stimulated respiration. These differences were not a result of any apparent differences in overall mitochondrial numbers between the two cell types (Figure 2C). Coincident with this decrease in mitochondrial oxygen consumption, we noted that Atg7-/- MEFs generated more lactic acid, consistent with an increase reliance on glycolysis (Figure 2D). This shift away from aerobic respiration and towards cytosolic glycolysis in Atg7-/- MEFs presumably represents a compensatory mechanism to maintain intracellular energetic homeostasis in the setting of dysfunctional mitochondria.


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)

Alterations in the energetics of Atg7 -/- MEFs.                                            (A) Western blot                                            analysis of wild type (+/+) or Atg7-/- MEFs for the expression                                            of Atg7, p62 and actin (loading control). (B) Measurement of oxygen                                            consumption for WT and Atg7-/-  MEFs under basal conditions,                                            following the addition of the mitochondrial electron chain inhibitor                                            oligomycin (0.5 μM), or in the presence of the mitochondrial uncoupler FCCP                                            (1 μM), to determine maximal oxidative capacity.  Shown is the average fold                                            change +/- SEM in oxygen consumption (WT MEFs basal respiration=1) obtained                                            from 5 experiments each performed in triplicate. (C)  Assessment of                                            mitochondrial number in WT or Atg7-/- MEFs. DNA was isolated                                            from WT (n=3 independent WT MEF cell isolates) and Atg7-/- MEFs (n=3                                            independent Atg7-/- MEF cell isolates) and quantitative PCR                                            analysis performed for the mitochondrial-encoded gene ND1 and the                                            nuclear-encoded gene H19.  (D) Relative extracellular acidification                                            rates indicating lactic acid production and hence glyolytic rates in WT or                                            Atg7-/- MEFs.  Shown is the average +/- SEM fold change in                                            lactic acid production from 8 experiments each performed in triplicate. *                                            p≤0.05; ** p≤0.01.
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Figure 2: Alterations in the energetics of Atg7 -/- MEFs. (A) Western blot analysis of wild type (+/+) or Atg7-/- MEFs for the expression of Atg7, p62 and actin (loading control). (B) Measurement of oxygen consumption for WT and Atg7-/- MEFs under basal conditions, following the addition of the mitochondrial electron chain inhibitor oligomycin (0.5 μM), or in the presence of the mitochondrial uncoupler FCCP (1 μM), to determine maximal oxidative capacity. Shown is the average fold change +/- SEM in oxygen consumption (WT MEFs basal respiration=1) obtained from 5 experiments each performed in triplicate. (C) Assessment of mitochondrial number in WT or Atg7-/- MEFs. DNA was isolated from WT (n=3 independent WT MEF cell isolates) and Atg7-/- MEFs (n=3 independent Atg7-/- MEF cell isolates) and quantitative PCR analysis performed for the mitochondrial-encoded gene ND1 and the nuclear-encoded gene H19. (D) Relative extracellular acidification rates indicating lactic acid production and hence glyolytic rates in WT or Atg7-/- MEFs. Shown is the average +/- SEM fold change in lactic acid production from 8 experiments each performed in triplicate. * p≤0.05; ** p≤0.01.
Mentions: In order to further characterize the defect in mitochondrial function within the context of intact cells, we next isolated mouse embryonic fibroblasts (MEFs) from wild type or Atg7-/- embryos (Figure 2A). Compared to WT MEFs, Atg7-/- MEFs exhibited a reduction in basal oxygen consumption (Figure 2B). In addition, we noted that Atg7-/- MEFs demonstrated a marked reduction in maximal mitochondrial oxidative capacity, as assessed by the levels of FCCP-stimulated respiration. These differences were not a result of any apparent differences in overall mitochondrial numbers between the two cell types (Figure 2C). Coincident with this decrease in mitochondrial oxygen consumption, we noted that Atg7-/- MEFs generated more lactic acid, consistent with an increase reliance on glycolysis (Figure 2D). This shift away from aerobic respiration and towards cytosolic glycolysis in Atg7-/- MEFs presumably represents a compensatory mechanism to maintain intracellular energetic homeostasis in the setting of dysfunctional mitochondria.

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