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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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Mice deficient in Atg7 expression within pancreatic β cells demonstrate altered mitochondria. (A) Western blot analysis of purified                                            pancreatic islets obtained from Atg7F/+:Rip2-CRE or Atg7F/F:Rip2-CRE                                            mice demonstrating the relative expression of Atg7, p62 and actin (loading                                            control). (B) Intracellular insulin levels (mean +/- SEM) in                                            pancreatic tissue of 8-9 week old Atg7F/+:Rip2-Cre (n=4 mice) or                                            Atg7F/F:Rip2-Cre mice (n=5 mice). The slight reduction in                                            insulin levels in the Atg7F/F:Rip2-Cre mice was not significant                                            when compared to the control. (C) Pancreatic sections of control                                            Atg7F/+:Rip2-Cre or Atg7F/F:Rip2-Cre mice were                                            stained for non-β cell components within the islets with the                                            simultaneous use of anti-glucagon, anti-somatostatin, and anti-polypeptide                                            antibodies. (D) Serial sections were used to visualize β cells                                            with an anti-insulin antibody.  Eight week old mice lacking autophagy in                                            β cells have qualitatively similar levels of α, δ, and polypeptide producing cells                                            within their islets, as well as similar levels of β cells when                                            compared to control mice. (E) Electron micrographs demonstrating the                                            accumulation of swollen, dysmorphic mitochondria within the Atg7-deficient                                            β cells. (F) Isolated islets from control and Atg7-/-                                            mice were assessed for fold +/- SEM changes in basal respiration (Atg7F/+:Rip2-Cre                                            isolated islets=1), and for oxygen consumption in the presence of                                            oligomycin (0.5 μM) or FCCP (0.5 μM). Results are normalized to islet                                            protein concentration and are from n=4 mice per genotype. (G)                                            Impaired glucose tolerance in Atg7F/F:Rip2-Cre mice. Blood                                            glucose measurements were made in 8-10 week-old control mice Atg7F/+:Rip2-Cre                                            (n=10 mice) or Atg7F/F:Rip2-Cre mice  (n=8 mice) following the                                            IP injection of D-glucose (1 g/kg).  Data represent the mean +/- SEM. *p≤0.05;                                            **p≤0.01.
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Figure 5: Mice deficient in Atg7 expression within pancreatic β cells demonstrate altered mitochondria. (A) Western blot analysis of purified pancreatic islets obtained from Atg7F/+:Rip2-CRE or Atg7F/F:Rip2-CRE mice demonstrating the relative expression of Atg7, p62 and actin (loading control). (B) Intracellular insulin levels (mean +/- SEM) in pancreatic tissue of 8-9 week old Atg7F/+:Rip2-Cre (n=4 mice) or Atg7F/F:Rip2-Cre mice (n=5 mice). The slight reduction in insulin levels in the Atg7F/F:Rip2-Cre mice was not significant when compared to the control. (C) Pancreatic sections of control Atg7F/+:Rip2-Cre or Atg7F/F:Rip2-Cre mice were stained for non-β cell components within the islets with the simultaneous use of anti-glucagon, anti-somatostatin, and anti-polypeptide antibodies. (D) Serial sections were used to visualize β cells with an anti-insulin antibody. Eight week old mice lacking autophagy in β cells have qualitatively similar levels of α, δ, and polypeptide producing cells within their islets, as well as similar levels of β cells when compared to control mice. (E) Electron micrographs demonstrating the accumulation of swollen, dysmorphic mitochondria within the Atg7-deficient β cells. (F) Isolated islets from control and Atg7-/- mice were assessed for fold +/- SEM changes in basal respiration (Atg7F/+:Rip2-Cre isolated islets=1), and for oxygen consumption in the presence of oligomycin (0.5 μM) or FCCP (0.5 μM). Results are normalized to islet protein concentration and are from n=4 mice per genotype. (G) Impaired glucose tolerance in Atg7F/F:Rip2-Cre mice. Blood glucose measurements were made in 8-10 week-old control mice Atg7F/+:Rip2-Cre (n=10 mice) or Atg7F/F:Rip2-Cre mice (n=8 mice) following the IP injection of D-glucose (1 g/kg). Data represent the mean +/- SEM. *p≤0.05; **p≤0.01.

Mentions: Given the profound alterations observed in isolated mitochondria derived from Atg7 deficient skeletal muscle and the observation that NAC treatment could at least partially reverse the metabolic defects observed in Atg7-/- MEFs, we next sought to assess whether these principles could be applied to the physiological defects seen in an in vivo model of Atg7 deficiency. Since our skeletal muscle conditional Atg7-/- mice did not exhibit an overt phenotype, we created an additional model in which Atg7 was deleted within pancreatic β cells by crossing the Atg7-floxed mice with RIP2-Cre animals. Western blot analysis from purified pancreatic islets demonstrated that conditional knockout animals (Atg7F/F:RIP2-Cre) had reduced or absent Atg7 expression and a corresponding increase in p62 levels (Figure 5A). In young mice, deletion of Atg7 within β cells did not result in significant alterations in pancreatic insulin expression (Figure 5B). Similarly, the cellular composition of individual pancreatic islets was largely unperturbed in 8 week old mice (Figure 5 C, D). In contrast, electron micrographs of control or knockout tissues revealed the early accumulation of markedly abnormal mitochondria within the β cells of Atg7 deficient mice (Figure 5E). Analysis of basal and FCCP-stimulated respiration from isolated pancreatic islets revealed a significant decrease in basal mitochondrial respiration and a marked decrease inmitochondrial oxidative capacity in Atg7 deficient islets (Figure 5F). Consistent with previous reports describing animals with alterations in β cell mitochondria [12,13,19], mice lacking Atg7 within their β cells also exhibited marked abnormalities in glucose tolerance (Figure 5G).


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)

Mice deficient in Atg7 expression within pancreatic β cells demonstrate altered mitochondria. (A) Western blot analysis of purified                                            pancreatic islets obtained from Atg7F/+:Rip2-CRE or Atg7F/F:Rip2-CRE                                            mice demonstrating the relative expression of Atg7, p62 and actin (loading                                            control). (B) Intracellular insulin levels (mean +/- SEM) in                                            pancreatic tissue of 8-9 week old Atg7F/+:Rip2-Cre (n=4 mice) or                                            Atg7F/F:Rip2-Cre mice (n=5 mice). The slight reduction in                                            insulin levels in the Atg7F/F:Rip2-Cre mice was not significant                                            when compared to the control. (C) Pancreatic sections of control                                            Atg7F/+:Rip2-Cre or Atg7F/F:Rip2-Cre mice were                                            stained for non-β cell components within the islets with the                                            simultaneous use of anti-glucagon, anti-somatostatin, and anti-polypeptide                                            antibodies. (D) Serial sections were used to visualize β cells                                            with an anti-insulin antibody.  Eight week old mice lacking autophagy in                                            β cells have qualitatively similar levels of α, δ, and polypeptide producing cells                                            within their islets, as well as similar levels of β cells when                                            compared to control mice. (E) Electron micrographs demonstrating the                                            accumulation of swollen, dysmorphic mitochondria within the Atg7-deficient                                            β cells. (F) Isolated islets from control and Atg7-/-                                            mice were assessed for fold +/- SEM changes in basal respiration (Atg7F/+:Rip2-Cre                                            isolated islets=1), and for oxygen consumption in the presence of                                            oligomycin (0.5 μM) or FCCP (0.5 μM). Results are normalized to islet                                            protein concentration and are from n=4 mice per genotype. (G)                                            Impaired glucose tolerance in Atg7F/F:Rip2-Cre mice. Blood                                            glucose measurements were made in 8-10 week-old control mice Atg7F/+:Rip2-Cre                                            (n=10 mice) or Atg7F/F:Rip2-Cre mice  (n=8 mice) following the                                            IP injection of D-glucose (1 g/kg).  Data represent the mean +/- SEM. *p≤0.05;                                            **p≤0.01.
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Figure 5: Mice deficient in Atg7 expression within pancreatic β cells demonstrate altered mitochondria. (A) Western blot analysis of purified pancreatic islets obtained from Atg7F/+:Rip2-CRE or Atg7F/F:Rip2-CRE mice demonstrating the relative expression of Atg7, p62 and actin (loading control). (B) Intracellular insulin levels (mean +/- SEM) in pancreatic tissue of 8-9 week old Atg7F/+:Rip2-Cre (n=4 mice) or Atg7F/F:Rip2-Cre mice (n=5 mice). The slight reduction in insulin levels in the Atg7F/F:Rip2-Cre mice was not significant when compared to the control. (C) Pancreatic sections of control Atg7F/+:Rip2-Cre or Atg7F/F:Rip2-Cre mice were stained for non-β cell components within the islets with the simultaneous use of anti-glucagon, anti-somatostatin, and anti-polypeptide antibodies. (D) Serial sections were used to visualize β cells with an anti-insulin antibody. Eight week old mice lacking autophagy in β cells have qualitatively similar levels of α, δ, and polypeptide producing cells within their islets, as well as similar levels of β cells when compared to control mice. (E) Electron micrographs demonstrating the accumulation of swollen, dysmorphic mitochondria within the Atg7-deficient β cells. (F) Isolated islets from control and Atg7-/- mice were assessed for fold +/- SEM changes in basal respiration (Atg7F/+:Rip2-Cre isolated islets=1), and for oxygen consumption in the presence of oligomycin (0.5 μM) or FCCP (0.5 μM). Results are normalized to islet protein concentration and are from n=4 mice per genotype. (G) Impaired glucose tolerance in Atg7F/F:Rip2-Cre mice. Blood glucose measurements were made in 8-10 week-old control mice Atg7F/+:Rip2-Cre (n=10 mice) or Atg7F/F:Rip2-Cre mice (n=8 mice) following the IP injection of D-glucose (1 g/kg). Data represent the mean +/- SEM. *p≤0.05; **p≤0.01.
Mentions: Given the profound alterations observed in isolated mitochondria derived from Atg7 deficient skeletal muscle and the observation that NAC treatment could at least partially reverse the metabolic defects observed in Atg7-/- MEFs, we next sought to assess whether these principles could be applied to the physiological defects seen in an in vivo model of Atg7 deficiency. Since our skeletal muscle conditional Atg7-/- mice did not exhibit an overt phenotype, we created an additional model in which Atg7 was deleted within pancreatic β cells by crossing the Atg7-floxed mice with RIP2-Cre animals. Western blot analysis from purified pancreatic islets demonstrated that conditional knockout animals (Atg7F/F:RIP2-Cre) had reduced or absent Atg7 expression and a corresponding increase in p62 levels (Figure 5A). In young mice, deletion of Atg7 within β cells did not result in significant alterations in pancreatic insulin expression (Figure 5B). Similarly, the cellular composition of individual pancreatic islets was largely unperturbed in 8 week old mice (Figure 5 C, D). In contrast, electron micrographs of control or knockout tissues revealed the early accumulation of markedly abnormal mitochondria within the β cells of Atg7 deficient mice (Figure 5E). Analysis of basal and FCCP-stimulated respiration from isolated pancreatic islets revealed a significant decrease in basal mitochondrial respiration and a marked decrease inmitochondrial oxidative capacity in Atg7 deficient islets (Figure 5F). Consistent with previous reports describing animals with alterations in β cell mitochondria [12,13,19], mice lacking Atg7 within their β cells also exhibited marked abnormalities in glucose tolerance (Figure 5G).

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