NAD؉ (nicotinamide adenine dinucleotide) is an essential cofactor involved in various biological processes including calorie restriction-mediated life span extension. Administration of nicotinamide riboside (NmR) has been shown to ameliorate deficiencies related to aberrant NAD ؉ metabolism in both yeast and mammalian cells. However, the biological role of endogenous NmR remains unclear. Here we demonstrate that salvaging endogenous NmR is an integral part of NAD ؉ metabolism. A balanced NmR salvage cycle is essential for calorie restrictioninduced life span extension and stress resistance in yeast. Our results also suggest that partitioning of the pyridine nucleotide flux between the classical salvage cycle and the NmR salvage branch might be modulated by the NAD ؉ -dependent Sir2 deacetylase. Furthermore, two novel deamidation steps leading to nicotinic acid mononucleotide and nicotinic acid riboside production are also uncovered that further underscore the complexity and flexibility of NAD ؉ metabolism. In addition, utilization of extracellular nicotinamide mononucleotide requires prior conversion to NmR mediated by a periplasmic phosphatase Pho5. Conversion to NmR may thus represent a strategy for the transport and assimilation of large nonpermeable NAD ؉ precursors. Together, our studies provide a molecular basis for how NAD ؉ homeostasis factors confer metabolic flexibility.

Nicotinamide adenine dinucleotide (NADNAD ϩ and its reduced form NADH are essential pyridine nucleotides mediating redox reactions in cellular metabolism. In addition, NAD ϩ is an essential substrate in several protein modification reactions, in particular sirtuin-mediated protein deacetylation and the addition of ADP-ribose moieties. These modifications are essential for the proteins functioning in Ca 2ϩ

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...

The Sir2 family proteins (sirtuins) are evolutionally conserved NAD + (nicotinamide adenine dinucleotide)-dependent protein deacetylases and ADP-ribosylases, which have been shown to play important roles in the regulation of stress response, gene transcription, cellular metabolism and longevity. Recent studies have also suggested that sirtuins are downstream targets of calorie restriction (CR), which mediate CR-induced beneficial effects including life span extension in a NAD + -dependent manner. CR extends life span in many species and has been shown to ameliorate many age-associated disorders such as diabetes and cancers. Understanding the mechanisms of CR as well as the regulation of sirtuins will therefore provide insights into the molecular basis of these age-associated metabolic diseases. This review focuses on discussing advances in studies of sirtuins and NAD + metabolism in genetically tractable model system, the budding yeast Saccharomyces cerevisiae. These studies have unraveled key metabolic longevity factors in the CR signaling and NAD + biosynthesis pathways, which may also contribute to the regulation of sirtuin activity. Many components of the NAD + biosynthesis pathway and CR signaling pathway are conserved in yeast and higher eukaryotes including humans. Therefore, these findings will help elucidate the mechanisms underlying age-associated metabolic disease and perhaps human aging.

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.