Calorie restriction (CR) extends the life span of numerous species, from yeast to rodents. Yeast Sir2 is a nicotinamide adenine dinucleotide (NAD+-dependent histone deacetylase that has been proposed to mediate the effects of CR. However, this hypothesis has been challenged by the observation that CR can extend yeast life span in the absence of Sir2. Here, we show that Sir2-independent life-span extension is mediated by Hst2, a Sir2 homolog that promotes the stability of repetitive ribosomal DNA, the same mechanism by which Sir2 extends life span. These findings demonstrate that the maintenance of DNA stability is critical for yeast life-span extension by CR and suggest that, in higher organisms, multiple members of the Sir2 family may regulate life span in response to diet.

Recent studies suggest that increased mitochondrial metabolism and the concomitant decrease in NADH levels mediate calorie restriction (CR)-induced life span extension. The mitochondrial inner membrane is impermeable to NAD (nicotinamide adenine dinucleotide, oxidized form) and NADH, and it is unclear how CR relays increased mitochondrial metabolism to multiple cellular pathways that reside in spatially distinct compartments. Here we show that the mitochondrial components of the malate-aspartate NADH shuttle (Mdh1 [malate dehydrogenase] and Aat1 [aspartate amino transferase]) and the glycerol-3-phosphate shuttle (Gut2, glycerol-3-phosphate dehydrogenase) are novel longevity factors in the CR pathway in yeast. Overexpressing Mdh1, Aat1, and Gut2 extend life span and do not synergize with CR. Mdh1 and Aat1 overexpressions require both respiration and the Sir2 family to extend life span. The mdh1⌬aat1⌬ double mutation blocks CR-mediated life span extension and also prevents the characteristic decrease in the NADH levels in the cytosolic/nuclear pool, suggesting that the malate-aspartate shuttle plays a major role in the activation of the downstream targets of CR such as Sir2. Overexpression of the NADH shuttles may also extend life span by increasing the metabolic fitness of the cells. Together, these data suggest that CR may extend life span and ameliorate age-associated metabolic diseases by activating components of the NADH shuttles.[Keywords: Calorie restriction (CR); Sir2; aging; NADH shuttles; respiration; metabolism] Supplemental material is available at http://www.genesdev.org.

Calorie restriction (CR) extends life span in a wide variety of species. Recent studies suggest that an increase in mitochondrial metabolism mediates CR-induced life span extension. Here we present evidence that Lat1 (dihydrolipoamide acetyltransferase), the E2 component of the mitochondrial pyruvate dehydrogenase complex, is a novel metabolic longevity factor in the CR pathway. Deleting the LAT1 gene abolishes life span extension induced by CR. Overexpressing Lat1 extends life span, and this life span extension is not further increased by CR. Similar to CR, life span extension by Lat1 overexpression largely requires mitochondrial respiration, indicating that mitochondrial metabolism plays an important role in CR. Interestingly, Lat1 overexpression does not require the Sir2 family to extend life span, suggesting that Lat1 mediates a branch of the CR pathway that functions in parallel to the Sir2 family. Lat1 is also a limiting longevity factor in nondividing cells in that overexpressing Lat1 extends cell survival during prolonged culture at stationary phase. Our studies suggest that Lat1 overexpression extends life span by increasing metabolic fitness of the cell. CR may therefore also extend life span and ameliorate age-associated diseases by increasing metabolic fitness through regulating central metabolic enzymes. Calorie restriction (CR)2 is the most effective intervention known to extend life span in a variety of species, including mammals (1, 2). CR has also been shown to delay the onset or reduce the incidence of many age-related diseases (1). Although it has been suggested that CR may work by reducing the levels of reactive oxygen species due to a slowing in metabolism (1, 3), the mechanism by which CR extends life span and ameliorates age-associated diseases is still uncertain.Moderate CR can be imposed in the budding yeast Saccharomyces cerevisiae by reducing the glucose concentration from 2 to 0.5% in rich media (4 -9). Under this CR condition, the growth rate remains robust, and yeast mother cells show an extended replicative life span (division potential) of about 20 -30%. Variations in CR protocols have been described where limitation of amino acids and other nutrients accompanies carbon source limitation (10, 11). These regimens may represent another possible longevity pathway that functions in parallel to CR in rich media. Genetic models of CR have also been identified and studied in multiple strain backgrounds, such as PSY316 (4, 5, 7), W303 (4, 8, 9, 12), and BY4742 (13). These CR mimics include a hexokinase mutant (hxk2⌬) and mutations that down-regulate the glucose-sensing cyclic-AMP/protein kinase A pathway: the temperature-sensitive alleles of the adenylate cyclase (cdc35-1) or the RAS nucleotide exchange protein (cdc25-10) and deletions of the glucose-sensing protein Gpa2 and Gpr1. Additional CR genetic models, the tor1⌬ and sch9⌬ mutants, have recently been reported to extend yeast life span (14, 15). The nutrient-sensing Target of Rapamycin (TOR) pathway and Sch9 kinase (a homolog of the...

Calorie restriction (CR) induces a metabolic shift towards mitochondrial respiration; however, molecular mechanisms underlying CR remain unclear. Recent studies suggest that CR-induced mitochondrial activity is associated with nitric oxide (NO) production. To understand the role of mitochondria in CR, we identify and study Saccharomyces cerevisiae mutants with increased NO levels as potential CR mimics. Analysis of the top 17 mutants demonstrates a correlation between increased NO, mitochondrial respiration, and longevity. Interestingly, treating yeast with NO donors such as GSNO (S-nitrosoglutathione) is sufficient to partially mimic CR to extend lifespan. CR-increased NO is largely dependent on mitochondrial electron transport and cytochrome c oxidase (COX). Although COX normally produces NO under hypoxic conditions, CR-treated yeast cells are able to produce NO under normoxic conditions. Our results suggest that CR may derepress some hypoxic genes for mitochondrial proteins that function to promote the production of NO and the extension of lifespan.