Endocrinol Metab.  2015 Mar;30(1):65-70. 10.3803/EnM.2015.30.1.65.

Mitochondrial Complexes I and II Are More Susceptible to Autophagy Deficiency in Mouse beta-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

• KMID: 2407106
• DOI: http://doi.org/10.3803/EnM.2015.30.1.65

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 beta-cells.
METHODS
To evaluate the effect of autophagy deficiency on mitochondrial function in pancreatic beta-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 beta-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 beta-TC6 cells also reduced the expression of complexes I and II, with marginal significance (P<0.1).
CONCLUSION
Impairment of autophagy in pancreatic beta-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.

Keyword

Autophagy; Insulin-secreting cells; Mitochondria; Mitochondrial complex

MeSH Terms

Adenosine
Adenosine Triphosphate
Animals
Antimycin A
Autophagy*
Down-Regulation
Glucose
Insulin-Secreting Cells
Mice*
Mitochondria
Oxygen Consumption
Real-Time Polymerase Chain Reaction
Respiration
RNA, Messenger
Adenosine
Adenosine Triphosphate
Antimycin A
Glucose
RNA, Messenger

Figure

• 

  Fig. 1 Basal oxygen consumption rates in islets isolated from Atg7Δβ-cell and control mice (n=5 to 8 per group). Data are presented as fold changes±SE, and comparisons were performed using a Mann-Whitney U test. Atg7, autophagy-related 7. aP<0.05 compared to the control.

• 

  Fig. 2 Intracellular adenosine 5'-triphosphate (ATP) content induced by glucose stimulation of islets isolated from Atg7Δβ-cell and control mice (n=3 to 10 per group). (A) ATP content under low-glucose (1.6 mM) and high-glucose (16 mM) conditions. All values were normalized relative to those of control islets under low glucose conditions. (B) Fold change in ATP content induced by high-glucose stimulation. Data are presented as fold changes±SE, and comparisons were performed using a Mann-Whitney U test. Atg7, autophagy-related 7; NS, not significant. aP<0.05 compared to control.

• 

  Fig. 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.

• 

  Fig. 4 Expression of mitochondrial complex genes by quantitative real-time polymerase chain reaction (A) in Atg7Δβ-cell and control islets (n=7 to 8 per group), and (B) in β-TC6 cells transfected with siAtg7 or with siNS as a control (n=4). The expression levels were normalized to that of glyceraldehyde 3-phosphate dehydrogenase. Data are presented as means±SE, and comparisons were performed using Student t tests or Mann-Whitney U tests, depending on sample variances. P value is presented above the bar graph. Atg7, autophagy-related 7. aP<0.05 compared to control islets.

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