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Originally published In Press as doi:10.1074/jbc.M607661200 on January 2, 2007

J. Biol. Chem., Vol. 282, Issue 9, 6161-6171, March 2, 2007
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The Dihydrolipoamide Acetyltransferase Is a Novel Metabolic Longevity Factor and Is Required for Calorie Restriction-mediated Life Span Extension*

Erin Easlon, Felicia Tsang, Ivanka Dilova, Chen Wang, Shu-Ping Lu, Craig Skinner, and Su-Ju Lin1

From the Section of Microbiology, University of California, Davis, California 95616

Received for publication, August 11, 2006 , and in revised form, December 11, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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{Delta}) 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{Delta} and sch9{Delta} 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 Akt kinase family) are known to interact with the protein kinase A pathway to regulate cell growth (16, 17).

Many studies have linked CR to a conserved longevity factor, Sir2 (4, 5, 18–22). In yeast, CR requires NAD (nicotinamide adenine dinucleotide, oxidized form) and Sir2 (4), a key regulator of life span in both yeast and animals (23, 24). Sir2 exhibits an NAD-dependent histone deacetylase activity that is conserved among the Sir2 family members (25–27) and is required for chromatin silencing and life span extension by CR (4, 5). It has been suggested that yeast Sir2 extends life span by increasing chromatin silencing at specific genomic loci, thereby decreasing genome instability and inappropriate gene expression (28). Yeast Sir2 may also increase the fitness of newborn cells by asymmetric partitioning of the oxidatively damaged proteins to mother cells (29). The Sir2 family in higher eukaryotes also plays an important role in CR-induced life span extension. Both worm and fly Sir2 homologs are required for life span extension in several CR models (19, 22).

In yeast, CR induces a shunting of carbon metabolism from fermentation to the mitochondrial tricarboxylic acid cycle (5). The concomitant increase in respiration is necessary and sufficient for the activation of Sir2-mediated silencing and extension in life span (5). The fact that respiration produces NAD from NADH (30, 31) as well as the finding that NADH can function as a competitive inhibitor of Sir2 activity (7) reinforce the idea that an increase in the NAD/NADH ratio activates Sir2 during CR. A link between CR and increased mitochondrial metabolism has also been reported in mammals. For example, in mice, CR was found to increase levels of endothelial nitricoxide synthase, mediating an increase in respiration and mitochondrial biogenesis (20). Concomitant with an increase in endothelial nitric-oxide synthase concentration, enhanced expression of the mammalian Sir2 ortholog Sirt1 was observed, thus reinforcing the link between respiration and Sir2 activation (20).

Other mechanisms for CR-induced Sir2-dependent life span extension demonstrate that nictotinamide, a by-product of NAD degradation in the Sir2-mediated deacetylation reaction (32), also affects Sir2 activity. Overexpression of the nicotinamidase, Pnc1, increases yeast life span and suppresses the inhibitory effect of nictotinamide on Sir2 deacetylase activity in vivo and in vitro (8, 33). Recently, a Sir2-independent CR pathway has been described (13). Two Sir2 family members, Hst2 and Hst1, have been suggested to play a role in this Sir2-independent pathway under certain CR conditions (9). It remains highly possible that CR also induces other proteins in addition to the Sir2 family to extend life span.

Many mitochondrial metabolic enzymes are regulated by the NAD/NADH ratio and are also likely to mediate CR-induced life span extension. To further understand the mechanism of CR, we characterized one such metabolic target of CR, the conserved NAD-dependent pyruvate dehydrogenase complex (PDC). The PDC acts in the mitochondria to convert pyruvate into acetyl-CoA (34), an activity regulated by the NAD/NADH ratio (35). In this study, we showed that a functional PDC is required for CR-induced life span extension. Overexpressing components of the PDC extended life span, suggesting that PDC is a limiting longevity factor. The role of mitochondrial metabolism in CR and several genetic models of CR are also discussed.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Strains and Media—Yeast strain BY4742 MAT{alpha} his3{Delta}1 leu2{Delta}0 lys2{Delta}0 ura3{Delta}0 was acquired from Open Biosystems and has been previously described (36). W303AR MATa ura3-1 leu2-3, 112 his3-11, 15 trp1-1 ade2-1 RDN1::ADE2 can1-100 has been described (23). Rich medium YPD and synthetic medium were made as described (37). Medium used for life span analysis was YEP (2% Bacto-peptone, 1% yeast extract, 1.5% agar) supplemented with filter-sterilized glucose at a final concentration of 2, 0.5, or 0.05%. All gene deletions in this study were generated in our laboratory by replacing the wild type genes with the reusable Kanr marker as described in Ref. 38 and verified by PCR using oligonucleotides flanking the genes of interest. The Lat1 overexpression construct pADH1-LAT1 was made as follows. A pair of oligonucleotides were designed to amplify the LAT1 coding region (from start codon to stop codon) via PCR. This pair of oligonucleotides also added a NotI site to the 5' end and a NheI site to the 3' end of the LAT1 gene. After PCR amplification, DNA was digested with NotI and NheI and then ligated to ppp81 digested with the same enzymes, resulting in pADH1-LAT1. The Lpd1 overexpression construct pADH1-LPD1 and the Pda1 overexpression construct pADH1-PDA1 were made the same way. Both constructs carried a functional LAT1 or LPD1 as verified by the ability to suppress the growth defects of the lat1{Delta} and lpd1{Delta} mutant, respectively, on nonfermentable carbon. The cdc25-10 mutants were made by two-step allele replacement as previously described (4). The Hap4-overexpressing construct pADH1-HAP4 has been previously described (5). Strains overexpressing Lat1, Lpd1, or Hap4 were made by integrating PacI-linearized pADH1-LAT1, pADH1-LPD1, or pADH1-HAP4 into the genomic ADH1 promoter. Strains overexpressing Pda1 were made by integrating NheI-linearized pADH1-PDA1 into the genomic PDA1 locus. Strains carrying control vectors were made by integrating PacI-linearized ppp81 into the genomic ADH1 promoter.

Replicative Life Span Analysis—All life span analyses in this study were carried out on YEP plates supplemented with different concentrations of glucose with 50 cells/strain/experiment as previously described (39) with a few modifications. Fresh growing cells were patched daily onto a fresh YEP plate with 2% glucose at least 3 days prior to life span analysis. Cells from frozen stocks were grown on YEP plates with 2% glucose (repatched daily) for at least 7 days prior to life span analysis. On the day of life span analysis, cells were repatched onto the side of the life span plates and then allowed to grow. After 2 h, 10 groups of cells were moved from the initial patch and arrayed onto the center of the life span plate (one strain/plate) using a micromanipulator and allowed to grow for 1.5 h (one or two divisions). Virgin cells were then selected and subjected to life span analysis. To reduce the chance of human errors and biases, life span assays were measured at least three times by two or three researchers.

NAD and NADH Measurements—Intracellular levels of the NAD and NADH nucleotides were determined as described previously (40) with a few modifications. In brief, cells were grown to an A600 of ~1, and then 107 cells were harvested in duplicates by centrifugation in two 1.5-ml tubes. Acid extraction was performed in one tube to obtain NAD, and alkali extraction was performed in the other to obtain NADH. 3 µl of neutralized cell extract (~105 cells) was used for the enzymatic cycling reaction as previously described (40). The concentration of nucleotides was measured fluorometrically with excitation at 365 nm and emission monitored at 460 nm. Standard curves for determining NAD and NADH concentrations were obtained as follows: NAD and NADH were added into the acid and alkali buffer to a final concentration of 0, 2.5, 5, and 7.5 µM, which were then treated with the same procedure along with other samples. The fluorometer was calibrated each time before use with 0, 5, 10, 20, 30, and 40 µM NADH to ensure that the detection was within a linear range.

Chronological Life Span Analysis—Three single colonies from each strain were analyzed in each experiment as previously described (41) with a few modifications. Cells were grown in synthetic medium supplemented with 2% glucose and four times of the auxotrophic amino acids (uracil, histidine, lysine, and leucine) at a starting A600 of 0.1. Cell viability was monitored daily by plating a fraction of the culture onto fresh medium to determine the colony-forming units. The rate of cell survival was calculated by normalizing the colony-forming units to the highest cell number (the stationary phase) for each strain.

RNA Extraction and Northern Blot Analysis—50 ml of cells were grown in YPD containing 2, 0.5, or 0.05% glucose as indicated to midlogarithmic phase (A600 of 0.5–0.6) and concentrated by centrifugation, and total RNA extraction was performed using a hot phenol method employing phase lock tubes (Eppendorf) as previously described (42). 20 µg of total RNA was loaded in each lane. After electrophoresis, the RNA was transferred to Duralon-UV membrane (Stratagene). The membranes were then probed, washed, exposed to a phosphorimaging screen, and analyzed using a STORM 860 imaging system and software provided by the manufacturer (Amersham Biosciences). DNA templates for the Northern probes were made as described previously (42). In short, PCR and specific gene primers were utilized to prepare the DNA templates using BY4742 genomic DNA. Northern probes were made using [{alpha}-32P]dCTP and Ready-to-Go DNA-labeling beads (Amersham Biosciences).

Ribosomal DNA (rDNA) Recombination—Rates of the rDNA recombination were determined by the rate of loss of ADE2 in the rDNA of strain W303AR at the first cell division (half-sectored colonies) as previously described (43). Cells were grown at 30 °C for 2 days, followed by 4–5 days at 4 °C for color enhancement. A total of more than 70,000 colonies were examined for each strain in each experiment.

Isolation and Immunofluorescence Microscopy Analysis of Young and Old Mother Cells—Yeast cells were labeled with EZ-link Sulfo-NHS-LC-biotin (Pierce), allowed to divide for ~7 generations, and then isolated with streptavidin-magnetic beads as previously described (39). "Young cells" refers to generation 0–1 cells that have not divided or have divided once only. "Old cells" refers to generation 6–7 cells that have divided 6–7 times. Strains for visualization of the mitochondrial structures were constructed by transformation with the pVT100-mtGFP plasmid carrying subunit 9 of the F0-ATPase of Neurospora crassa and a URA3 marker (44). Cells recovered from magnetic bead sorting were washed with phosphate-buffered saline (pH 7.2), stained with calcofluor/fluorescent brightener (for bud scars) or 4',6-diamidino-2-phenylindole (for DNA) for 5 min, washed again with phosphate-buffered saline, and then examined under a Nikon Eclipse 80i fluorescence microscope with a Hamamatsu ORCA-ER camera at x1000 magnification.

Pyruvate Dehydrogenase Complex Activity Assay—Activity of the PDC was assayed in mitochondrial lysates as described (35). Briefly, cells were grown in YPD to an A600 of 1–2, and then the crude mitochondrial fraction (pellet P12000) was purified as described (45). Enzyme activity was assayed immediately after preparation of mitochondrial extracts. The assay mixture contained 100 mM potassium phosphate buffer, pH 7.4, 1 mM CaCl2, 1 mM MgCl2, 2.5 mM NAD, 2 mM thiamine pyrophosphate, 2 mM cysteine, 5 mM pyruvate. The reaction was started by the addition of coenzyme A (final concentration 0.15 mM), and the increase of NADH concentration was followed at 340 nm. PDC activity was calculated and shown as the amount of NADH produced/µg of mitochondrial proteins/h (nmol·µg–1·h–1).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
CR induces an increase in mitochondrial metabolic activity (5, 20). The PDC catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA, the key substrate entering the mitochondrial tricarboxylic acid cycle. The PDC is composed of multiple copies of three separate enzymes: pyruvate dehydrogenase (E1, ~20 copies), dihydrolipoamide acetyltransferase (E2, ~60 copies), and lipoamide dehydrogenase (E3, ~6 copies) (46). In yeast, the E1 component of the PDC is composed of two different subunits encoded by the PDA1 and PDB1 gene, respectively (34). The activity of E1 is regulated by reversible phosphorylation catalyzed by the pyruvate dehydrogenase kinases (PDKs) and pyruvate dehydrogenase phosphate phosphatases. Phosphorylation of E1 by PDK leads to PDC inactivation (47–50). To date, four PDK isoforms and two pyruvate dehydrogenase phosphate phosphatase isoforms have been identified in mammals, whereas in yeast, only one PDK isoform (Pkp1) and one pyruvate dehydrogenase phosphate phosphatase isoform (Ppp1) have been identified (49). The E2 component, encoded by the LAT1 gene is the core of the PDC and plays both an enzymatic and structural role (51). The E3 component, encoded by the LPD1 gene, is also a component in the 2-oxoglutarate dehydrogenase and glycine decarboxylase complexes (34). The protein X (encoded by the PDX1 gene) plays a structural role in the PDC; it binds and positions E3 to the E2 core (52). Deleting any genes in PDC resulted in defects in generating acetyl-CoA (34). To examine whether PDC is a novel metabolic target of CR, we first determined whether a functional PDC was required for CR-induced life span extension. Replicative life span (RLS) of cells devoid of Lat1, the core component of the PDC, was analyzed under 0.5% glucose-induced CR conditions as previously described (4–9). As shown in Fig. 1A, deleting the LAT1 gene abolished CR-induced life span extension, suggesting that a functional PDC was required for CR. We then determined whether components of the PDC were indeed limiting longevity factors. As shown in Fig. 1B, cells overexpressing the E2 component, Lat1, showed a ~30% increase in average life span. Interestingly, overexpressing the E1 or E3 components, Pda1 and Lpd1, did not extend life span (Fig. 1B). Deleting the recently discovered yeast PDK, Pkp1 (49) also failed to extend life span (Fig. 1B). These results suggested that Lat1 was the limiting longevity factor in the PDC. We then examined whether Lat1 overexpression functioned as a CR mimic to extend life span. As shown in Fig. 1C, Lat1 overexpression did not synergize with 0.5% glucose to extend life span, suggesting that Lat1 functions in the same pathway as CR to extend life span.

The Sir2 family members play important roles in CR in several model systems (4, 5, 18–22). In yeast, 0.5% glucose requires the Sir2 family to mediate life span extension (4, 5, 9). To determine whether Lat1 overexpression functioned through the Sir2 family to extend life span, we overexpressed Lat1 in the sir2{Delta}fob1{Delta}hst1{Delta}hst2{Delta} quadruple mutant, a genetic model for studying the requirement of the Sir2 family for life span (9). Interestingly, deletions of the Sir2 family did not abolish the long life span induced by Lat1 overexpression (Fig. 2A). For comparison, mutations in the Sir2 family largely abolished 0.5% induced life span extension (Fig. 2B, left). As a further test, we determined whether Lat1 affected genome stability at the rDNA loci. It has been suggested that CR and the Sir2 family extend life span by increasing chromatin silencing at the rDNA loci, thereby decreasing the production of toxic rDNA circles (28). Interestingly, Lat1 overexpression slightly decreased the recombination rate at the rDNA loci, whereas the lat1{Delta} mutation slightly increased the recombination rate (Fig. 2C). These studies suggested that Lat1 functioned at least in part by increasing the stability at the rDNA loci. Lat1 may thus mediate a branch of the CR pathway that functioned in parallel to the Sir2 family.


Figure 1
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  		FIGURE 1.
The role of Lat1 in CR. A, 0.5% glucose-induced CR requires Lat1 to extend replicative life span. Shown is life span analysis of the wild type cells and the lat1{Delta} mutant cells grown in 2 and 0.5% glucose. B, Lat1 overexpression extends replicative life span. Life span analysis of the wild type cells carrying a control vector, the Lat1 overexpression plasmid, the Lpd1 overexpression plasmid, the Pda1 overexpression plasmid, and the pkp1{Delta} mutant. C, Lat1 overexpression does not synergize with 0.5% glucose to extend replicative life span. Shown is life span analysis of the wild type and Lat1-overexpressing (Lat1-oe) cells grown under 2 and 0.5% glucose. D, 0.05% glucose-induced CR requires Lat1 to extend replicative life span. Life span analysis of the wild type cells and the lat1{Delta} mutant cells grown in 2 and 0.05% glucose. E, Lat1 overexpression does not synergize with 0.05% glucose to extend replicative life span. Life span analysis of the wild type and Lat1-overexpressing cells grown under 2 and 0.05% glucose. F, Northern blot analysis of the Lat1 mRNA levels in cells grown in 2, 0.5, and 0.05% glucose CR conditions. WT, BY4742 wild type. One set of representative data is shown.

 
A Sir2-independent CR pathway induced by 0.05% glucose (13, 15, 53), a significant reduction in glucose concentration compared with 0.5% glucose-induced CR (4–9), has recently been described. We therefore examined whether Lat1 also functioned in this pathway. Similar to Lat1 overexpression (Fig. 2A), 0.05% glucose induced ~30% life span extension in the Sir2 family mutants (Fig. 2B, right). It was also noteworthy that although 0.05% glucose extended life span effectively in the Sir2 family mutants, it appeared to be a less effective CR condition (~10% extension) in wild type cells compared with 0.5% glucose (~30% extension) (Fig. 2B, right and left) and Lat1 overexpression (~30% extension) (Fig. 1B). It is possible that under 0.05% glucose, the homolog of the mammalian AMP-activated protein kinase Snf1 becomes activated, leading to reduced replicative life span (54). To examine whether Lat1 played a role in this CR pathway, we first analyzed whether Lat1 was required for 0.05% glucose-induced life span extension. As shown in Fig. 1D, 0.05% glucose did not significantly extend life span in the lat1{Delta} mutant. We then examined whether 0.05% would further synergize with Lat1 overexpression to extend life span. Interestingly, 0.05% glucose abolished the life span extension induced by Lat1 overexpression (Fig. 1E), suggesting that Lat1 overexpression and 0.05% glucose exerted opposite effects on certain common downstream targets. Alternatively, 0.05% glucose might reduce Lat11 overexpression back to the wild type level. We therefore determined the Lat1 mRNA level in cells overexpressing Lat1 grown under different CR conditions. As shown in Fig. 1F, 0.05% glucose did not reduce Lat1 expression to wild type levels (~5-fold increase in Lat1 expression). Interestingly, 0.05% glucose induced a ~1.5-fold increase in the Lat1 mRNA levels, whereas 0.5% glucose had no affect on the Lat1 mRNA levels. It was therefore very likely that Lat1 overexpression, 0.5% glucose, and 0.05% glucose induced CR function through distinct yet overlapping mechanisms.

We then determined whether CR and Lat1 overexpression increased PDC activity. Interestingly, as shown in Table 1, there was no significant increase in steady state PDC activity in cells grown under CR conditions or overexpressing Lat1. It was possible that our assay was not sensitive enough to detect subtle differences. These results also suggested that CR and Lat1 overexpression extended life span by continuous replacement of damaged E2 subunits in the PDC to maintain PDC activity over time rather than increase overall steady state PDC activity. Alternatively but not mutually exclusively, components of the PDC are damaged over time, and some of these damages may decrease the ability of PDC to respond to changes in the NAD/NADH ratio. CR and Lat1 overexpression may alleviate these problems by changing the NAD/NADH ratio or by increasing the sensitivity of PDC to the NAD/NADH ratio. It has been suggested that CR extends life span by increasing the intracellular NAD/NADH ratio (7). To further understand how Lat1 overexpression extended life span and the interaction between Lat1 overexpression and CR, we determined whether Lat1 played a role in the intracellular NAD/NADH homeostasis. As shown in Fig. 3A, Lat1 overexpression did not affect either NAD or NADH levels. For comparison and consistent with previous studies, 0.5% glucose increased the NAD/NADH ratio by decreasing the NADH level ~2-fold (Fig. 3B). Interestingly, 0.05%, a more stringent CR condition, did not further increase the NAD/NADH ratio (Fig. 3B). In fact, as the glucose concentration further decreased to 0.05%, the NADH level was increased to a level close to that in non-CR cells (Fig. 3B). Therefore, 0.05% glucose might have induced a distinct metabolic state compared with 0.5% glucose-mediated CR. We also examined whether Lat1 was required for the 0.5% glucose CR-induced increase in the NAD/NADH ratio. As shown in Fig. 3C, 0.5% was able to elicit an increase in the NAD/NADH ratio in both wild type and the lat1{Delta} mutant cells. These results demonstrated that Lat1 did not affect the steady state level of NAD and NADH. Therefore, it seemed unlikely that Lat1 overexpression extended life span by regulating the NAD/NADH ratio.


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  		TABLE 1
PDC enzymatic activity in various strains

PDC activity is shown as the amount of NADH produced/µg of mitochondrial proteins/h (nmol·µg-1·h-1). Results show one representative set of three independent experiments, each conducted in triplicate.

 


Figure 2
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  		FIGURE 2.
Role of the Sir2 family in CR- and Lat1 overexpression-induced replicative life span extension. A, Lat1 overexpression does not require the Sir2 family to extend life span. Shown is life span analysis of the wild type, Lat1-overexpressing (Lat1-oe) cells, the fob1{Delta}sir2{Delta}hst1{Delta}hst2{Delta} quadruple mutant, and the fob1{Delta}sir2{Delta}hst1{Delta}hst2{Delta} mutant with Lat1 overexpression on 2% glucose medium. One set of representative data is shown. B, 0.5% glucose but not 0.05% glucose requires the Sir2 family to extend life span. The percentage increase in life span (relative to 2% glucose) induced by 0.5% glucose (left) and by 0.05% glucose (right) is shown. Shown is life span analysis of the wild type and the fob1{Delta}sir2{Delta}hst1{Delta}hst2{Delta} quadruple mutant cells grown in 0.05, 0.5, and 2% glucose. The results show the average of three independent experiments. Error bars, S.D. values. C, Lat1 regulates recombination at the rDNA loci. Shown are measurements of the rate of loss of an ADE2 marker integrated at the rDNA in wild-type, Lat1-overexpressing (left), and lat1{Delta} (right) mutant cells. These results show the average of three independent experiments, each conducted in quadruplicate. Error bars, S.D. values. WT, BY4742 wild type with (A and C, left) or without (B and C, right) a control vector.

 
Mitochondrial respiration has been shown to be required for life span extension induced by certain CR conditions (5, 53). We therefore examined whether Lat1 overexpression also required functional mitochondrial respiration using the cyt1{Delta} mutant. Cells carrying the cyt1{Delta} mutation (devoid of cytochrome c1) are defected in electron transport chain activities (55). Similar to 0.5% glucose (Fig. 4B), Lat1 overexpression required a functional respiratory chain to elicit maximum life span extension (Fig. 4A). Interestingly, the cyt1{Delta} mutant showed a ~30% reduction in life span compared with wild type cells grown in 2% glucose (Fig. 4B), suggesting that respiration not only was required for maximum life span extension induced by Lat1 overexpression but also important for maintaining normal life span. Interestingly, both 0.5% and Lat1 overexpression appeared to restore the short life span of the cyt1{Delta} mutant back to that of the wild type cells (Fig. 4A), suggesting that 0.5% glucose and Lat1 overexpression were able to rescue certain growth defects associated with the cyt1{Delta} mutation. Consistent with previous reports (53), 0.05% glucose did not require functional mitochondrial respiration (Fig. 4B). In fact, 0.05% glucose extended life span much more efficiently in the cyt1{Delta} mutant (~50% increase) than in the wild type cells (~10% increase) (Fig. 4B), suggesting that 0.05% glucose may extend life span through an alternative pathway that is normally repressed by respiration.

We also analyzed the requirement of Cyt1 and Lat1 in several genetic models of CR to further examine the role of mitochondrial metabolism in CR. As shown in Fig. 5, we found that all CR genetic mimics tested required a functional respiratory chain for maximum life span extension. The hexokinase hxk2{Delta} mutant normally showed a 60–80% increase in life span (9, 13) (Fig. 5A). The cyt1{Delta} mutation largely reduced the life span of the hxk2{Delta} to almost wild type level (Fig. 5A). We also analyzed three additional CR mimics: the cdc25-10 (RAS GTPase) mutant, Hap4 overexpression, and the sch9{Delta} mutant. In the cdc25-10 mutant, the glucose-sensing cAMP-activated protein kinase A activity is down-regulated, thereby increasing life span (4). Hap4 is a component of the Hap complex that binds to the consensus sequence CCAAT and increases the transcription of many genes involved in mitochondrial metabolism (56). Overexpressing Hap4 has been shown to induce a metabolic shift toward respiration and extend life span in the PSY316 strain (5). Sch9 is a homolog of the Akt kinase family and is shown to extend life span (14, 15). As shown in Fig. 5, B–D, all of these CR genetic mimics extended life span significantly, and, as expected, Cyt1 was required for maximum life span extension. Similar to 0.5% glucose, most CR mimics also rescued the short life span of the cyt1{Delta} mutant to wild type level, suggesting that a respiration-independent pathway, which played a minor role, was also induced by CR to extend life span. In summary, these results demonstrated that respiration played an important role in CR (0.5% glucose and most genetic mimics of CR, including Lat1 overexpression) and 0.05% glucose represented a different pathway of CR. We then examined whether Lat1 was required for life span extension in these genetic models of CR. As shown in Fig. 6, B and C, the lat1{Delta} mutation significantly reduced the life span extension by Hap4 overexpression and the cdc25-10 mutation, suggesting that Lat1 played an important role in these two CR genetic models. Interestingly, Lat1 did not seem to affect the long life span of the hxk2{Delta} (Fig. 6A) and sch9{Delta} (Fig. 6D) mutants, suggesting that the hxk2{Delta} and sch9{Delta} mutants acted through a Lat1-independent pathway. This Lat1-independent pathway was, however, dependent on a functional respiratory chain, since life span extension by the hxk2{Delta} and sch9{Delta} mutations was largely abolished in the cyt1{Delta} mutant.


Figure 3
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  		FIGURE 3.
Role of Lat1 and CR in the regulation of intracellular NAD/NADH levels. Shown are measurements of the intracellular NAD and NADH levels in wild type, Lat1-overexpressing (Lat1-oe), and lat1{Delta} mutant cells grown in 2%, 0.5%, and 0.05% glucose. A, Lat1 overexpression does not increase the NAD/NADH ratio. B, levels of the NAD and NADH of cells grown in different glucose concentrations. 0.5% glucose induces a ~2-fold increase in the NAD/NADH ratio, whereas 0.05% glucose does not elicit the same effect. C, the lat1{Delta} mutation does not affect the NAD/NADH levels in cells under 0.5% glucose-induced CR. One representative set of three independent experiments, each conducted in quadruplicate, is shown. Error bars, S.D. values. WT, BY4742 wild type.

 
The lat1{Delta} mutation has been shown to cause slow growth on nonfermentable carbon sources (34) and is synthetic lethal with deletions in the Hsp82 gene, a member of the Hsp90 chaperones (57). The lat1{Delta} mutation may therefore decrease metabolic fitness of the cell, thereby preventing the benefits of CR. Consistent with this hypothesis, we found that the lat1{Delta} mutants gave rise to more petite daughter cells when a single cell lineage was followed. Fig. 7A (left) showed the frequencies of petite formation in cells derived from single colonies directly from the culture plates after 2-day growth. The lat1{Delta} mutant exhibited a 5-fold increase in petite formation compared with wild type cells. Interestingly, this phenotype was not observed in fresh growing cells (Day 1) compared with the 2-day-old cell population. These results indicated that Lat1 was essential for maintaining metabolic fitness in cells during prolonged culture. The lat1{Delta} mutant was therefore likely to reduce the chronological life span of cells. In yeast, "chronological life span (CLS)" refers to the length of time cells remain viable in stationary phase or postdiauxic phase (14). The lat1{Delta} mutant indeed showed a significant reduction in viability in the 2-day-old culture (Fig. 7A, right); only 35% (normal + petite) of the lat1{Delta} mutant cells remained viable (Fig. 7A). Although we failed to detect significant decreases in petite formation by Lat1 overexpression (data not shown), it was very likely that our assay was not sensitive enough to detect small differences. Therefore, we directly examined the CLS of cells overexpressing Lat1. As shown in Fig. 7B (left), Lat1 overexpression significantly extended the survival of cells grown in stationary phase, indicating that Lat1 was a limiting longevity factor in both dividing and nondividing cells. For comparison, we analyzed cells overexpressing Pda1 and Lpd1 and cells lacking Pkp1. Interestingly, Lpd1 overexpression also extended CLS (Fig. 7B, right), whereas overexpression of Pda1 or deletion of Pkp1 did not significantly increase CLS (data not shown). These results suggested that, similar to Lat1, Lpd1 was also a limiting longevity factor in nondividing cells. Lpd1 overexpression, however, failed to extend replicative life span (Fig. 1B). This was probably due to the pleiotrophic effects induced by Lpd1 overexpression, since Lpd1 was also a component in the 2-oxoglutarate dehydrogenase and glycine decarboxylase complexes (34). CLS has been associated with an increase in stress resistance (14). However, our studies showed that neither Lat1 overexpression nor the lat1{Delta} mutation conferred resistance or sensitivity to paraquat, a chemical generating superoxide anions (data not shown), reinforcing the idea that Lat1 increased CLS by increasing metabolic fitness of the cells. This Lat1-mediated metabolic fitness also played a role in replicative life span regulation. As shown in Fig. 7C, yeast cells exhibited age-dependent increases in mitochondrial fission. In young cells, mitochondria appeared as dynamic tube-like structures (58) (Fig. 7C), whereas in old cells (6–7 generations old), mitochondria became highly fragmented (Fig. 7C). It has been shown that components regulating mitochondrial fusion and fission also regulate apoptosis (58). Shifting the balance toward fusion renders cells resistant to apoptotic stimuli, whereas blocking the fusion processes increases mitochondrial fragmentation and renders cells sensitive to apoptotic stimuli (58). The age-dependent mitochondrial fission phenotypes observed in old cells suggested that apoptosis may also play a role in replicative senescence. Interestingly, at least 10–20% of the old cells overexpressing Lat1 retained the tube-like structure, suggesting that Lat1 may increase life span by delaying age-dependent mitochondrial structural changes.


Figure 4
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  		FIGURE 4.
Role of mitochondrial respiration in CR- and Lat1 overexpression-induced replicative life span extension. A, Lat1 requires Cyt1 for life span extension. Shown is life span analysis of the wild type cells, the cyt1{Delta} mutant, and the cyt1{Delta} mutant with Lat1-overexpressing (Lat1-oe) cells grown in 2% glucose. WT, BY4742 wild type. One set of representative data is shown. B, 0.5% glucose fails to elicit maximum life span extension in the respiratory deficient cyt1{Delta} mutant, whereas 0.05% confers robust life span extension in this mutant. Shown is life span analysis of the wild type and cyt1{Delta} cells grown in 2, 0.5, and 0.05% glucose. The results show mean life span (generations) derived from three independent experiments. Error bars, S.D. values.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this report, we characterized the core (E2) component of the PDC, Lat1, as a novel longevity regulator required for CR-mediated life span extension. Overexpression of Lat1 extended life span and did not synergize with CR, whereas deletion of Lat1 abolished life span extension induced by CR and several genetic mimics of CR. Here we provide evidence that the central metabolic enzyme-Lat1/PDC functions at the interface of multiple longevity-regulating pathways (Fig. 8A). PDC converts pyruvate to acetyl-CoA, which initiates the mitochondrial tricarboxylic acid cycle, leading to the synthesis of metabolic intermediates for amino acid metabolism and energy production by respiration (34). As expected, Lat1 overexpression required mitochondrial respiration for life span extension. Most CR models examined in this study also required mitochondrial respiration for maximum life span extension, indicating that mitochondrial metabolism plays an important role in multiple CR pathways with one interesting exception, the 0.05% glucose-induced CR (Fig. 8A). Several lines of evidence suggest that 0.05% glucose- and 0.5% glucose-induced CR probably function through distinct pathways. In contrast to 0.5% glucose, 0.05% glucose did not require mitochondrial respiration or the Sir2 family to extend life span. In fact, 0.05% glucose extended life span more effectively in the Sir2 family mutants and the cyt1{Delta} mutants than in wild type cells. These results suggest that the 0.05% glucose pathway is normally repressed by components in the 0.5% glucose pathway (Fig. 8A). It remains unclear how 0.05% glucose functions to extend life span. Although Lat1 appeared to play an important role in the 0.05% glucose CR pathway (0.05% glucose-induced Lat1 expression, and the lat1{Delta} mutation abolished 0.05% glucose-induced life span extension) certain differences exist between these two pathways. For example, Lat1 overexpression required a functional respiratory chain to elicit life span extension, whereas 0.05% glucose did not.

Both 0.5% glucose CR and the Sir2 family have been suggested to extend life span by increasing the genome stability at the rDNA loci (28). Lat1 appeared to affect rDNA recombination; Lat1 overexpression decreased the recombination rate, whereas the lat1{Delta} mutation increased the recombination rate without a significant reduction in replicative life span. Perhaps the increase in rDNA recombination caused by the lat1{Delta} mutation is relatively minor (less than 2-fold) compared with the sir2{Delta} mutation, which increases rDNA recombination by at least 10-fold and shortens replicative life span (23). These results suggested that Lat1 did not extend life span solely by repressing rDNA recombination. Interestingly, overexpressing Lat1 did not require the Sir2 family to extend life span, suggesting that Lat1 overexpression functioned in parallel to the Sir2 family to mediate CR. Alternatively, the Sir2 family may also extend life span by regulating metabolic enzymes. In fact, the Sir2 family has recently been connected to the synthesis of acetyl-CoA production through deacetylation of acetyl-CoA synthetase (59, 60). The Sir2 homolog in Salmonella enerica, CobB, also activates acetyl-CoA synthetase by NAD-dependent deacetylation (61). Acetyl-CoA synthetase produces acetyl-CoA from acetate, acting as a bypass for acetyl-CoA production in the absence of a functional PDC. Although acetyl-CoA synthetase is not the major pathway for generating acetyl-CoA, it is still possible that certain CR conditions may act through acetyl-CoA synthetase. Moreover, in addition to being a major source of biological energy, acetyl-CoA is also used by the cell as a substrate for protein modification and regulation of gene transcription (62–64). For example, transcription factors whose activities are dramatically affected by acetylation status, such as TFIIB, have higher levels of acetylation in mice after fasting, correlating with proposed increases in acetyl-CoA levels (64). These studies suggest that certain metabolic factors, such as acetyl-CoA, directly connect metabolism, metabolic stress, and perhaps CR to transcriptional regulation. Together, these studies demonstrate that CR is mediated by multiple pathways, which are interconnected by different longevity factors.


Figure 5
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  		FIGURE 5.
Respiration is required for maximum replicative life span extension in genetic models of CR. A, the hxk2{Delta} mutation requires Cyt1 for maximum life span extension. Shown is life span analysis of the wild type cells, the hxk2{Delta} and cyt1{Delta} mutants, and the hxk2{Delta}cyt1{Delta} double mutant cells on 2% glucose medium. B, overexpression of Hap4 increases life span and requires Cyt1 for maximum extension. Shown is life span analysis of the wild type and Hap4-overexpressing (Hap4-oe) cells, the cyt1{Delta} mutant, and the cyt1{Delta} mutant with Hap4 overexpression on 2% glucose media. C, the cdc25-10 mutation increases life span of the BY4742 strain and requires Cyt1 for maximum extension. Shown is life span analysis of the wild type and cdc25-10 mutant cells, the cyt1{Delta} mutant, and the cdc25-10 cyt1{Delta} double mutant cells on 2% glucose medium. D, the sch9{Delta} mutation requires Cyt1 for maximum life span extension. Shown is life span analysis of the wild type and sch9{Delta} mutant cells, the cyt1{Delta} mutant, and the sch9{Delta}cyt1{Delta} double mutant cells on 2% glucose medium. WT, BY4742 wild type. One set of representative data is shown.

 
Components of the PDC are susceptible to age-related oxidative damage (65, 66). This selective damage to the PDC over time may cause aging and certain age-associated diseases. In fact, several age-associated diseases have been linked to defects in the PDC (67, 68). Since Lat1 overexpression and CR do not increase the steady state PDC activity (Table 1), it is very likely that Lat1 overexpression and CR extend life span by continuous replacement of damaged E2 subunits in the PDC (Fig. 8B). It would be interesting to examine whether Lat1 overexpression and CR maintain PDC activity in old cells. CR may also increase life span by decreasing age-induced damage to the PDC, since CR has been shown to decrease the level of reactive oxygen species and prevent specific age-induced damages to metabolic enzymes (3, 69, 70). In addition, CR (0.5% glucose) may activate PDC activity by increasing the NAD/NADH ratio. The E1 and E3 components of the PDC have been shown to be subject to NAD/NADH regulation (35, 47–50). In higher eukaryotes, it has been suggested that the PDC activity is mainly controlled through E1. The activity of E1 is inhibited by reversible phosphorylation catalyzed by E1-specific kinases (PDKs) and phosphatases. The activity of PDK is activated by NADH and inhibited by NAD (48, 50). In prokaryotes, the control of PDC activity is mainly through E3. The activity of E3 in the PDC requires NAD (as an electron acceptor) and is inhibited by NADH. In yeast, both E1 and E3 seem to be the controlling points of PDC activity (35, 49, 71). The E2 component does not appear to be a direct target of NAD/NADH regulation. E2 may, however, affect the ability of E1 and E3 to interact with NAD and/or NADH. It has been suggested that binding of E3 to the E2 core increases the NADH sensitivity of E3 (72). Furthermore, the mammalian PDKs (E1 kinases) are known to associate with PDC through binding to the lipoyl domains of E2. Interestingly, the yeast PDK (Pkp1) lacks the tail domain found in mammalian PDKs that binds to the lipoyl domain of E2. Therefore, it remains unclear how Pkp1 interacts with PDC in yeast. Collectively, these data suggest that E2 plays a key role in regulating PDC activity and that overexpressing E2 may extend life span by increasing the sensitivity of PDC to the NAD/NADH ratio.


Figure 6
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  		FIGURE 6.
Role of Lat1 in genetic models of CR. A, the hxk2{Delta} mutation does not require Lat1 for life span extension. Replicative life span analysis of the wild type cells, the hxk2{Delta} and lat1{Delta} mutants, and the hxk2{Delta}lat1{Delta} double mutant cells on 2% glucose media. B, overexpression of Hap4 requires Lat1 for maximum life span extension. Shown is replicative life span analysis of the wild type and Hap4-overexpressing (Hap4-oe) cells, the lat1{Delta} mutant, and the lat1{Delta} mutant with Hap4 overexpression on 2% glucose medium. C, the cdc25-10 mutation requires Lat1 for life span extension. Shown is replicative life span analysis of the wild type and cdc25-10 mutant cells, the lat1{Delta} mutant, and the cdc25-10 lat1{Delta} double mutant cells on 2% glucose medium. D, the sch9{Delta} mutation does not require Lat1 for life span extension. Replicative life span analysis of the wild type and sch9{Delta} mutant cells, the cyt1{Delta} mutant, and the sch9{Delta}cyt1{Delta} double mutant cells on 2% glucose medium. WT, BY4742 wild type. One set of representative data is shown.

 
Other CR conditions may extend life span by increasing the expression levels of Lat1 (such as 0.05% glucose) or by PDC-independent pathways (the sch9{Delta} and hxk2{Delta} mutants). It was noteworthy that the hxk2{Delta} and sch9{Delta} long lived mutants did not require Lat1 for life span extension. Although these two mutants may extend life span by a mechanism independent of Lat1/PDC, both mutants required mitochondrial respiration for life span extension, suggesting that mitochondrial metabolism played an important role in these mutants. It was therefore likely that the acetyl-CoA bypass pathway (mediated by the pyruvate decarboxylase and acetyl-CoA synthetase etc.) (34) was induced in both mutants to generate sufficient acetyl-CoA for life span extension. This bypass may also account for the normal life span seen in the lat1{Delta} mutant strains. Interestingly, growing cells on rich medium containing acetate as the sole carbon source severely reduced the life span (an average of 3–5 divisions; data not shown), suggesting that the PDC bypass was unable to adequately produce life-sustaining levels of acetyl-CoA and that the PDC activity was essential for normal life span.

Overexpressing Lat1 may enhance metabolic fitness of the cell. Although overexpressing Lat1 did not increase resistance to oxidative stress, it did extend cell survival in stationary phase, a relatively stressful growth condition. In addition, we found that the lat1{Delta} mutant gave rise to more petite daughter cells, suggesting a decrease in metabolic fitness in these cells. It was noteworthy that although the lat1{Delta} mutants showed reduced chronological life span, they exhibited normal replicative life span. It is possible that the lat1{Delta} mutation primarily reduces the fitness of the daughter cells and therefore does not affect the division potential of the mother cells in which RLS was determined. Alternatively, since the lat1{Delta} mutation abolished CR-induced life span extension, it was very likely that the lat1{Delta} mutation also reduced the fitness of the mother cell. In this case, the lat1{Delta} mutant mother cells may maintain a normal life span and fitness at the expense of the fitness of the daughter cells. CLS has been suggested to be a model for studying senescence of postmitotic cells, whereas RLS resembles the proliferative potential of mitotic cells. The complex mechanisms underlying CLS and RLS regulation and whether these two pathways share common components are currently unclear. Although certain CR conditions appeared to extend both RLS and CLS (14), it has also been shown that RLS and CLS functioned through distinct downstream targets (14). Our studies demonstrated that components of the PDC played an important role in both types of life span regulation and were therefore likely to be conserved longevity regulators that functioned at the interface of these two pathways. Interestingly, feeding lipoic acid/lipoamide (an essential cofactor for E2 activity) to old rats alleviates several age-associated phenotypes and improves metabolic function (73). The activity of E2 may also play an important role in life span regulation in mammals. Treating yeast cells with subtoxic doses of lipoamide, however, does not extend yeast replicative life span (data not shown), further suggesting that E2 is the limiting factor in yeast. These data also suggest that the limiting components in PDC for life span extension are likely to vary in different species.

PDC is also an emerging metabolic target in cancer. Inactivation of PDC has been shown to be a critical step in adaptation to hypoxia-induced metabolic switches. In mammals, the activity of the E1{alpha} subunit of the PDC is regulated through phosphorylation by pyruvate dehydrogenase kinase, PDK1 (48). Upon E1 phosphorylation, the PDC becomes inactive and prevents pyruvate metabolism through the tricarboxylic acid cycle (48). Under hypoxic conditions, the hypoxia-inducible factor 1, HIF-1, up-regulates the transcription of PDK1 (74, 75). Inactivation of the PDC by PDK1 causes a shift from mitochondrial respiration to glycolysis, the main form of metabolism in 60–90% of tumor cells (76, 77). Together, these studies suggest that CR may prevent or delay the onset of cancer by activating the PDC.


Figure 7
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  		FIGURE 7.
Lat1 affects metabolic fitness of cells. A, the lat1{Delta} mutants exhibit decreased mitochondrial fitness and viability after prolonged culture. For each strain, 1000 cells derived from 1-day-old cell patches or 2-day-old single colonies were plated onto YPD plates. Colony-forming units were determined after 3 days. Left, the percentage of petite colonies; right, the percentage of colonies grown to normal size (both derived from the same experiment). One representative set of two independent experiments, each conducted in quadruplicate, is shown. Error bars, S.D. values. B, Lat1 and Lpd1 overexpression extends chronological life span. Fractions of viable wild type, Lat1-overexpressing (Lat1-oe), and Lpd1-overexpressing (Lpd1-oe) cells were determined from cultures grown to stationary phase. One representative set of three independent experiments, each conducted in triplicate, is shown. Error bars, S.D. values. C, Lat1 overexpression delays age-dependent changes in mitochondrial structure. Green fluorescent protein (GFP) shows the localization of the mitochondrial inner membrane F0-ATPase; 4',6-diamidino-2-phenylindole (DAPI) shows nuclear and mitochondrial DNA. One set of representative data is shown. WT, BY4742 wild type with (B), or without (A and C) a control vector.

 


Figure 8
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  		FIGURE 8.
Proposed models for the role of the PDC in multiple longevity pathways. A, multiple CR pathways act to affect longevity in yeast. Respiration is required for 0.5% CR and CR mimic-induced life span extension but inhibits the life span benefits of 0.05% CR. A functional PDC is required for some CR mimics and may be acting at the interface between 0.5 and 0.05% CR pathways. The PDC maintains metabolic fitness of the cell and functions in parallel to the Sir2 family to mediate CR effects. B, overexpressing the E2 component of the PDC may lead to increased metabolic fitness through continuous replacement of damaged subunits over time. Different CR conditions activate the PDC by different mechanisms. For example, 0.5% glucose-induced CR may increase metabolic fitness through the prevention of age-associated damages to the PDC and/or through the regulation of the NAD/NADH ratio. The 0.05% glucose-induced CR may act partially through increasing the expression level of E2. Furthermore, 0.5% glucose-induced CR may inhibit PDK by increasing the NAD/NADH ratio.

 

   FOOTNOTES
 
* This work was supported by NIA, National Institutes of Health, and the Ellison Medical Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 A new research scholar of the Ellison Medical Foundation. To whom correspondence should be addressed: Section of Microbiology, University of California, 323 Briggs Hall, One Shields Ave, Davis, CA 95616. Tel.: 530-754-6082; Fax: 530-752-9014; E-mail: slin{at}ucdavis.edu.

2 The abbreviations used are: CR, calorie restriction; PDC, pyruvate dehydrogenase complex; PDK, pyruvate dehydrogenase kinase; RLS, replicative life span; rDNA, ribosomal DNA; CLS, chronological life span. Back


   ACKNOWLEDGMENTS
 
We thank members of the Lin laboratory for discussions and suggestions, Dr. J. Nunnari for providing the pVT100-mtGFP plasmid, and Dr. B. Ames for suggestions.



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 DISCUSSION
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