Mitochondrial DNA copy number was also compared in the MEF cultures at passage 2. A qPCR analysis using primer sets targeting either the mitochondrial or nuclear genome indicated that MEFs derived from the IGF-I deficient mice contained a higher mitochondrial DNA content than the control MEFs. We provide evidence that autophagy occurs in quiescent cells even when sufficient nutrients are available and that inhibition of autophagy through IGF-I signaling can lead to the accumulation of cells with dysfunctional mitochondria and decreased long-term viability. Rapamycin, which enhances autophagy, can ameliorate the effects of IGF-I while inhibition of autophagy recapitulates some of these effects. Furthermore, it appears that a reduction in IGF-I in mice leads to enhanced autophagy and a similar decrease in depolarized mitochondria. Interestingly, this is accompanied by an increase in total mitochondrial mass. In total, the results indicate that inhibition of autophagy by IGF-I decreases cell viability through interference with mitochondrial turnover. These observations suggest that increased IGF-I signaling over long periods have unanticipated consequences that are distinct from the pro survival effects observed in acute settings. Autophagy has been identified as a process for the turnover of intracellular components which can be negatively regulated through the intracellular signaling pathway associated with mTOR. It involves a series of lysine-linked conjugation steps analogous to the ubiquitin conjugation required for proteasome targeting. This process is important for proper cellular function and defects in autophagy have been linked to several types of degenerative diseases. Although direct evidence that autophagy can influence longevity in mammals is lacking, experimental evidence suggests that an enhanced rate of autophagy during aging enhances liver function and appropriate levels of autophagy are essential for cardiac function. Autophagy has been linked to aging, and a reduction in autophagy during aging has been observed in rodents and other organisms. Caloric restriction increases autophagy in rodents, and genetic studies in Caenorhabditis elegans indicate that autophagy may be required for life-span extension by caloric restriction. Autophagy also increases during dauer formation in C. elegans and is required for life-span extension in daf-2 mutants. Genetic studies in C. elegans have found that autophagy genes are required for life-span extension in response to mutations in the insulin/insulin-like growth factor receptor and to mutations that induce caloric restriction, although there may be caveats to this connection that have not been fully appreciated since other studies indicate that suppression of autophagy in the adult may extend life span. Autophagy is an important mechanism for the clearance of mitochondria following damage and IGF-I has been Everolimus reported to influence this process but the relative importance of mitochondrial clearance under physiologic conditions is less clear.