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6 mM) and high-glucose (16 mM) conditions. ... Mitochondrial respiration 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 Bioactive Compound high throughput screening experiment, a marked reduction in oxygen consumption rate was observed in Atg7����-cell islets compared to controls (Plearn more 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. Fig. 3 Mitochondrial respiration in islets isolated from Atg7����-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 ... Expression of mitochondrial complex genes We examined the expression of mitochondrial respiratory complex genes in isolated islets and in ��-TC6 cells. In Atg7����-cell islets, expression of Atg7 mRNA was reduced by half (Fig. 4A). Among mitochondrial complexes, mitochondrial complex IV gene expression predominated. The expressions of mitochondrial complexes except complex IV were significantly lower in Atg7����-cell islets than in controls (PChloramben complex genes by quantitative real-time polymerase chain reaction (A) in Atg7����-cell and control islets (n=7 to 8 per group), and (B) in ��-TC6 cells transfected with siAtg7 or with siNS as a control ... DISCUSSION In pancreatic ��-cells, as in other mammalian cells, damaged mitochondria are removed by autophagy [12]. Impairment of autophagy is thus expected to result in the accumulation of damaged mitochondria, and we have previously reported abnormal swollen mitochondria in Atg7����-cell mice [7]. In this study, we observed the decreased oxygen consumption rate and ATP content, noted previously in autophagy-deficient pancreatic ��-cells [8,9]. We also report that impaired autophagy affects specific steps in the mitochondrial electron transfer system. Although Atg7����-cell islets showed a significant overall decrease in mitochondrial respiration compared to control islets (Fig.