Increased Life Span due to Calorie Restriction in Respiratory-Deficient Yeast

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Abstract
A model for replicative life span extension by calorie restriction (CR) in yeast has been proposed whereby reduced glucose in the growth medium leads to activation of the NAD+–dependent histone deacetylase Sir2. One mechanism proposed for this putative activation of Sir2 is that CR enhances the rate of respiration, in turn leading to altered levels of NAD+ or NADH, and ultimately resulting in enhanced Sir2 activity. An alternative mechanism has been proposed in which CR decreases levels of the Sir2 inhibitor nicotinamide through increased expression of the gene coding for nicotinamidase, PNC1. We have previously reported that life span extension by CR is not dependent on Sir2 in the long-lived BY4742 strain background. Here we have determined the requirement for respiration and the effect of nicotinamide levels on life span extension by CR. We find that CR confers robust life span extension in respiratory-deficient cells independent of strain background, and moreover, suppresses the premature mortality associated with loss of mitochondrial DNA in the short-lived PSY316 strain. Addition of nicotinamide to the medium dramatically shortens the life span of wild type cells, due to inhibition of Sir2. However, even in cells lacking both Sir2 and the replication fork block protein Fob1, nicotinamide partially prevents life span extension by CR. These findings (1) demonstrate that respiration is not required for the longevity benefits of CR in yeast, (2) show that nicotinamide inhibits life span extension by CR through a Sir2-independent mechanism, and (3) suggest that CR acts through a conserved, Sir2-independent mechanism in both PSY316 and BY4742. Calorie restriction slows aging and increases life span in nearly every organism studied. The mechanism by which this occurs is one of the most important unanswered questions in biogerontology. One popular theory, based on work from the budding yeast Saccharomyces cerevisiae, proposes that calorie restriction works by causing a metabolic shift toward increased mitochondrial respiration, resulting in activation of a family of proteins known as Sirtuins. This study demonstrates that life span extension by calorie restriction does not require respiration and occurs even in cells completely lacking mitochondrial DNA. Interestingly, calorie restriction protects yeast cells against a severe longevity defect associated with absence of mitochondrial DNA, suggesting the possibility that the consequences of age-associated mitochondrial dysfunction might be alleviated or prevented by calorie restriction.