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Mitochondrial Complexes I and II Are More Susceptible to Autophagy Deficiency in Mouse ╬▓-Cells.

Kim MJ, Choi OK, Chae KS, Kim MK, Kim JH, Komatsu M, Tanaka K, Lee H, Chung SS, Kwak SH, Cho YM, Park KS, Jung HS - Endocrinol Metab (Seoul) (2014)

Bottom Line: By Oxygraph-2k analysis, mitochondrial respiration in Atg7-deficient islets was significantly decreased overall, although state 3 respiration and responses to antimycin A were unaffected.The mRNA levels of mitochondrial complexes I, II, III, and V in Atg7-deficient islets were significantly lower than in control islets (P<0.05).Down-regulation of Atg7 in β-TC6 cells also reduced the expression of complexes I and II, with marginal significance (P<0.1).

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Seoul National University College of Medicine, Korea.; Department of Internal Medicine, Korea Cancer Center Hospital, Korea.

ABSTRACT

Background: Damaged mitochondria are removed by autophagy. Therefore, impairment of autophagy induces the accumulation of damaged mitochondria and mitochondrial dysfunction in most mammalian cells. Here, we investigated mitochondrial function and the expression of mitochondrial complexes in autophagy-related 7 (Atg7)-deficient ╬▓-cells.

Methods: To evaluate the effect of autophagy deficiency on mitochondrial function in pancreatic ╬▓-cells, we isolated islets from Atg7(F/F):RIP-Cre+ mice and wild-type littermates. Oxygen consumption rate and intracellular adenosine 5'-triphosphate (ATP) content were measured. The expression of mitochondrial complex genes in Atg7-deficient islets and in ╬▓-TC6 cells transfected with siAtg7 was measured by quantitative real-time polymerase chain reaction.

Results: Baseline oxygen consumption rate of Atg7-deficient islets was significantly lower than that of control islets (P<0.05). Intracellular ATP content of Atg7-deficient islets during glucose stimulation was also significantly lower than that of control islets (P<0.05). By Oxygraph-2k analysis, mitochondrial respiration in Atg7-deficient islets was significantly decreased overall, although state 3 respiration and responses to antimycin A were unaffected. The mRNA levels of mitochondrial complexes I, II, III, and V in Atg7-deficient islets were significantly lower than in control islets (P<0.05). Down-regulation of Atg7 in ╬▓-TC6 cells also reduced the expression of complexes I and II, with marginal significance (P<0.1).

Conclusion: Impairment of autophagy in pancreatic ╬▓-cells suppressed the expression of some mitochondrial respiratory complexes, and may contribute to mitochondrial dysfunction. Among the complexes, I and II seem to be most vulnerable to autophagy deficiency.

No MeSH data available.


Related in: MedlinePlus

Mitochondrial respiration in islets isolated from Atg7Δβ-cell and control mice (n=9 to 10 per group). (A) Representative graphs of mitochondrial respiration using Oxygraph-2k. Red lines indicate oxygen consumption rate in response to sequential loading of mitochondrial effectors (indicated by arrows below the graphs). (B) Quantitative comparisons of oxygen consumption rate in response to effectors. Data are presented as means±SE, and comparisons were performed using repeated measures analysis of variance and Mann-Whitney U tests. G+M, glutamate and malate; ADP, adenosine diphosphate; FCCP, carbonyl-cyanide-4-(trifluoromethoxy)-phenylhydrazone; AA, antimycin A; Atg7, autophagy-related 7. aP<0.05 compared to control islets.
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Figure 3: Mitochondrial respiration in islets isolated from Atg7Δβ-cell and control mice (n=9 to 10 per group). (A) Representative graphs of mitochondrial respiration using Oxygraph-2k. Red lines indicate oxygen consumption rate in response to sequential loading of mitochondrial effectors (indicated by arrows below the graphs). (B) Quantitative comparisons of oxygen consumption rate in response to effectors. Data are presented as means±SE, and comparisons were performed using repeated measures analysis of variance and Mann-Whitney U tests. G+M, glutamate and malate; ADP, adenosine diphosphate; FCCP, carbonyl-cyanide-4-(trifluoromethoxy)-phenylhydrazone; AA, antimycin A; Atg7, autophagy-related 7. aP<0.05 compared to control islets.

Mentions: It is unclear whether mitochondrial dysfunction reflects only the accumulation of damaged mitochondria or if there are other, direct effects on mitochondrial function. To determine whether specific respiratory states are influenced, we performed Oxygraph-2k analyses with isolated islets. Representative graphs are presented in Fig. 3A. Throughout the experiment, a marked reduction in oxygen consumption rate was observed in Atg7Δβ-cell islets compared to controls (P<0.01 by repeated measures analysis of variance) (Fig. 3B). Inhibitory effects of succinate (an inhibitor of complex II) and rotenone (an inhibitor of complex I) were relatively prominent, while there was no difference in responses to ADP (stage 3 respiration) or to antimycin A (an inhibitor of complex III). These findings suggest that complexes I and II are most vulnerable to autophagy deficiency.


Mitochondrial Complexes I and II Are More Susceptible to Autophagy Deficiency in Mouse ╬▓-Cells.

Kim MJ, Choi OK, Chae KS, Kim MK, Kim JH, Komatsu M, Tanaka K, Lee H, Chung SS, Kwak SH, Cho YM, Park KS, Jung HS - Endocrinol Metab (Seoul) (2014)

Mitochondrial respiration in islets isolated from Atg7Δβ-cell and control mice (n=9 to 10 per group). (A) Representative graphs of mitochondrial respiration using Oxygraph-2k. Red lines indicate oxygen consumption rate in response to sequential loading of mitochondrial effectors (indicated by arrows below the graphs). (B) Quantitative comparisons of oxygen consumption rate in response to effectors. Data are presented as means±SE, and comparisons were performed using repeated measures analysis of variance and Mann-Whitney U tests. G+M, glutamate and malate; ADP, adenosine diphosphate; FCCP, carbonyl-cyanide-4-(trifluoromethoxy)-phenylhydrazone; AA, antimycin A; Atg7, autophagy-related 7. aP<0.05 compared to control islets.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4384673&req=5

Figure 3: Mitochondrial respiration in islets isolated from Atg7Δβ-cell and control mice (n=9 to 10 per group). (A) Representative graphs of mitochondrial respiration using Oxygraph-2k. Red lines indicate oxygen consumption rate in response to sequential loading of mitochondrial effectors (indicated by arrows below the graphs). (B) Quantitative comparisons of oxygen consumption rate in response to effectors. Data are presented as means±SE, and comparisons were performed using repeated measures analysis of variance and Mann-Whitney U tests. G+M, glutamate and malate; ADP, adenosine diphosphate; FCCP, carbonyl-cyanide-4-(trifluoromethoxy)-phenylhydrazone; AA, antimycin A; Atg7, autophagy-related 7. aP<0.05 compared to control islets.
Mentions: It is unclear whether mitochondrial dysfunction reflects only the accumulation of damaged mitochondria or if there are other, direct effects on mitochondrial function. To determine whether specific respiratory states are influenced, we performed Oxygraph-2k analyses with isolated islets. Representative graphs are presented in Fig. 3A. Throughout the experiment, a marked reduction in oxygen consumption rate was observed in Atg7Δβ-cell islets compared to controls (P<0.01 by repeated measures analysis of variance) (Fig. 3B). Inhibitory effects of succinate (an inhibitor of complex II) and rotenone (an inhibitor of complex I) were relatively prominent, while there was no difference in responses to ADP (stage 3 respiration) or to antimycin A (an inhibitor of complex III). These findings suggest that complexes I and II are most vulnerable to autophagy deficiency.

Bottom Line: By Oxygraph-2k analysis, mitochondrial respiration in Atg7-deficient islets was significantly decreased overall, although state 3 respiration and responses to antimycin A were unaffected.The mRNA levels of mitochondrial complexes I, II, III, and V in Atg7-deficient islets were significantly lower than in control islets (P<0.05).Down-regulation of Atg7 in β-TC6 cells also reduced the expression of complexes I and II, with marginal significance (P<0.1).

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Seoul National University College of Medicine, Korea.; Department of Internal Medicine, Korea Cancer Center Hospital, Korea.

ABSTRACT

Background: Damaged mitochondria are removed by autophagy. Therefore, impairment of autophagy induces the accumulation of damaged mitochondria and mitochondrial dysfunction in most mammalian cells. Here, we investigated mitochondrial function and the expression of mitochondrial complexes in autophagy-related 7 (Atg7)-deficient ╬▓-cells.

Methods: To evaluate the effect of autophagy deficiency on mitochondrial function in pancreatic ╬▓-cells, we isolated islets from Atg7(F/F):RIP-Cre+ mice and wild-type littermates. Oxygen consumption rate and intracellular adenosine 5'-triphosphate (ATP) content were measured. The expression of mitochondrial complex genes in Atg7-deficient islets and in ╬▓-TC6 cells transfected with siAtg7 was measured by quantitative real-time polymerase chain reaction.

Results: Baseline oxygen consumption rate of Atg7-deficient islets was significantly lower than that of control islets (P<0.05). Intracellular ATP content of Atg7-deficient islets during glucose stimulation was also significantly lower than that of control islets (P<0.05). By Oxygraph-2k analysis, mitochondrial respiration in Atg7-deficient islets was significantly decreased overall, although state 3 respiration and responses to antimycin A were unaffected. The mRNA levels of mitochondrial complexes I, II, III, and V in Atg7-deficient islets were significantly lower than in control islets (P<0.05). Down-regulation of Atg7 in ╬▓-TC6 cells also reduced the expression of complexes I and II, with marginal significance (P<0.1).

Conclusion: Impairment of autophagy in pancreatic ╬▓-cells suppressed the expression of some mitochondrial respiratory complexes, and may contribute to mitochondrial dysfunction. Among the complexes, I and II seem to be most vulnerable to autophagy deficiency.

No MeSH data available.


Related in: MedlinePlus