Calorie restriction reduces rDNA recombination independently of rDNA silencing

Calorie restriction (CR) extends lifespan in yeast, worms, flies and mammals, suggesting that it acts via a conserved mechanism. In yeast, activation of the NAD‐dependent histone deacetylase, Sir2, by CR is thought to increase silencing at the ribosomal DNA, thereby reducing the recombination‐induced generation of extrachromosomal rDNA circles, hence increasing replicative lifespan. Although accumulation of extrachromosomal rDNA circles is specific to yeast aging, it is thought that Sirtuin activation represents a conserved longevity mechanism through which the beneficial effects of CR are mediated in various species. We show here that growing yeast on 0.05 or 0.5% glucose (severe and moderate CR, respectively) does not increase silencing at either sub‐telomeric or rDNA loci compared with standard (2% glucose) media. Furthermore, rDNA silencing was unaffected in the hxk2Δ, sch9Δ and tor1Δ genetic mimics of CR, but inhibited by FOB1 deletion. All these interventions extend lifespan in multiple yeast backgrounds, revealing a poor correlation between rDNA silencing and longevity. In contrast, CR and deletion of the FOB1, HXK2, SCH9 and TOR1 genes, all significantly reduced rDNA recombination. This silencing‐independent mechanism for suppressing rDNA recombination may therefore contribute to CR‐mediated lifespan extension.     

Calorie restriction alters mitochondrial protein acetylation

Calorie restriction (CR) increases lifespan in organisms ranging from budding yeast through mammals. Mitochondrial adaptation represents a key component of the response to CR. Molecular mechanisms underlying this adaptation are largely unknown. Here we show that lysine acetylation of mitochondrial proteins is altered during CR in a tissue-specific fashion. Via large-scale mass spectrometry screening, we identify 72 candidate proteins involved in a variety of metabolic pathways with altered acetylation during CR. Mitochondrial acetylation changes may play an important role in the pro-longevity CR response.     

Role for Sit4p-dependent mitochondrial dysfunction in mediating the shortened chronological lifespan and oxidative stress sensitivity of Isc1p-deficient cells

Saccharomyces cerevisiae cells lacking Isc1p, an orthologue of mammalian neutral sphingomyelinase 2, display a shortened lifespan and an increased sensitivity to oxidative stress. A lipidomic analysis revealed specific changes in sphingolipids that accompanied the premature ageing of Isc1p-deficient cells under severe calorie restriction conditions, including a decrease of dihydrosphingosine levels and an increase of dihydro-C(26) -ceramide and phyto-C(26) -ceramide levels, the latter raising the possibility of activation of ceramide-dependent protein phosphatases. Consequently, deletion of the SIT4 gene, which encodes for the catalytic subunit of type 2A ceramide-activated protein phosphatase in yeast, abolished the premature ageing and hydrogen peroxide sensitivity of isc1Δ cells. SIT4 deletion also abolished the respiratory defects and catalase A deficiency exhibited by isc1Δ mutants. These results are consistent with catabolic derepression associated with the loss of Sit4p. The overall results show that Isc1p is an upstream regulator of Sit4p and implicate Sit4p activation in mitochondrial dysfunction leading to the shortened chronological lifespan and oxidative stress sensitivity of isc1Δ mutants.     

Metabolic Depression and Increased Reactive Oxygen Species Production by Isolated Mitochondria at Moderately Lower Temperatures

Temperature (T) reduction increases lifespan, but the mechanisms are not understood. Because reactive oxygen species (ROS) contribute to aging, we hypothesized that lowering T might decrease mitochondrial ROS production. We measured respiratory response and ROS production in isolated mitochondria at 32, 35, and 37 °C. Lowering T decreased the rates of resting (state 4) and phosphorylating (state 3) respiration phases. Surprisingly, this respiratory slowdown was associated with an increase of ROS production and hydrogen peroxide release and with elevation of the mitochondrial membrane potential, ΔΨ_m. We also found that at lower T mitochondria produced more carbon-centered lipid radicals, a species known to activate uncoupling proteins. These data indicate that reduced mitochondrial ROS production is not one of the mechanisms mediating lifespan extension at lower T. They suggest instead that increased ROS leakage may mediate mitochondrial responses to hypothermia.     

Astrocytic metabolic and inflammatory changes as a function of age

This study examines age‐dependent metabolic‐inflammatory axis in primary astrocytes isolated from brain cortices of 7‐, 13‐, and 18‐month‐old Sprague–Dawley male rats. Astrocytes showed an age‐dependent increase in mitochondrial oxidative metabolism respiring on glucose and/or pyruvate substrates; this increase in mitochondrial oxidative metabolism was accompanied by increases in COX3/18SrDNA values, thus suggesting an enhanced mitochondrial biogenesis. Enhanced mitochondrial respiration in astrocytes limits the substrate supply from astrocytes to neurons; this may be viewed as an adaptive mechanism to altered cellular inflammatory–redox environment with age. These metabolic changes were associated with an age‐dependent increase in hydrogen peroxide generation (largely ascribed to an enhanced expression of NOX2) and NFκB signaling in the cytosol as well as its translocation to the nucleus. Astrocytes also displayed augmented responses with age to inflammatory cytokines, IL‐1β, and TNFα. Activation of NFκB signaling resulted in increased expression of nitric oxide synthase 2 (inducible nitric oxide synthase), leading to elevated nitric oxide production. IL‐1β and TNFα treatment stimulated mitochondrial oxidative metabolism and mitochondrial biogenesis in astrocytes. It may be surmised that increased mitochondrial aerobic metabolism and inflammatory responses are interconnected and support the functionality switch of astrocytes, from neurotrophic to neurotoxic with age.     

Dopamine modifies oxygen consumption and mitochondrial membrane potential in striatal mitochondria

Dopamine is a neurotransmitter that has been related to mitochondrial dysfunction. In this study, striatal intact mitochondria and submitochondrial membranes were incubated with different dopamine concentrations, and changes on mitochondrial function, hydrogen peroxide, and nitric oxide production were evaluated. A 35% decrease in state 3 oxygen uptake (active respiration state) was found after 1 mM dopamine incubation. In addition, mitochondrial respiratory control significantly decreased, indicating mitochondrial dysfunction. High dopamine concentrations induced mitochondrial depolarization. Also, evaluation of hydrogen peroxide production by intact striatal mitochondria showed a significant increase after 0.5 and 1 mM dopamine incubation. Incubation with 0.5 and 1 mM dopamine increased nitric oxide production in submitochondrial membranes by 28 and 49%, respectively, as compared with control values. This study provides evidence that high dopamine concentrations induce striatal mitochondrial dysfunction through a decrease in mitochondrial respiratory control and loss of membrane potential, probably mediated by free radical production.     

Sirtuin-independent effects of nicotinamide on lifespan extension from calorie restriction in yeast

Two models have been proposed for how calorie restriction (CR) enhances replicative longevity in yeast: (i) suppression of rDNA recombination through activation of the sirtuin protein deacetylase Sir2 or (ii) decreased activity of the nutrient-responsive kinases Sch9 and TOR. We report here that CR increases lifespan independently of all Sir2-family proteins in yeast. Furthermore, we demonstrate that nicotinamide, an inhibitor of Sir2-mediated deacetylation, interferes with lifespan extension from CR, but does so independent of Sir2, Hst1, Hst2, and Hst4. We also find that 5 mm nicotinamide, a concentration sufficient to inhibit other sirtuins, does not phenocopy deletion of HST3. Thus, we propose that lifespan extension by CR is independent of sirtuins and that nicotinamide has sirtuin-independent effects on lifespan extension by CR.     

Sirtuin在热量限制延长寿命机制中的研究进展

热量限制(caloric restriction, CR)可以引起细胞、生物体寿命延长和降低衰老相关疾病的发生,其中Sirtuin起着关键作用．Sirtuin将机体能量代谢和基因表达调控相偶联,通过赖氨酸去乙酰化改变蛋白质的活性和稳定性,从而调节衰老进程．酵母中度CR影响其复制寿命和时序寿命,主要依赖于激活Sir2,增加细胞内NAD+/NADH的比例和调节尼克酰胺浓度来实现．类似的机制也存在于秀丽线虫和果蝇中．哺乳动物在CR条件下SIRT1蛋白表达应答性上升,细胞中NAM磷酸基转移酶能够直接影响NAM和NAD+浓度,并影响SIRT1活性．NO表达增加能导致SIRT1上调和线粒体合成增加．SIRT1可能通过改变组蛋白、p53、NES1、FOXO等底物蛋白的乙酰化影响到细胞和个体的衰老．表明不同生物体中的Sirtuin及其同源类似物在CR条件下对衰老进程和寿命都起着非常重要的作用．     

Metformin-ROS-Nrf2 connection in the host defense mechanism against oxidative stress,apoptosis, cancers,and ageing

Reactive oxygen species (ROS) acts as a second messenger to trigger biological responses in low concentrations, while it is implicated to be toxic to biomolecules in high concentrations. Mild inhibition of respiratory chain Complex I by metformin at physiologically relevant concentrations stimulates production of low-level mitochondrial ROS. The ROS seems to induce anti-oxidative stress response via activation of nuclear factor erythroid 2-related factor 2 (Nrf2) and glutathione peroxidase (GPx), which results in not only elimination of ROS but also activation of cellular responses including resistance to apoptosis, metabolic changes, cell proliferation, senescence prevention, lifespan extension, and immune T cell activation against cancers, regardless of its effect controlling blood glucose level and T2DM. Although metformin's effect against T2DM, cancers, and ageing, are believed mostly attributed to the activation of AMP-activated protein kinase (AMPK), the cellular responses involving metformin-ROS-Nrf2 axis might be another natural asset to improve healthspan and lifespan.     

Oxidative stress regulates the interaction of p16 with Cdk4

Oxidative stress can have a myriad of effects on many different cell types. The mechanisms by which these effects occur are not completely known. Chimeric proteins of the GAL4 DNA binding domain and Cdk4, or the GAL4 activation domain with p16, were expressed in the yeast two-hybrid system. Cells expressing these chimeric proteins were cultured with hydrogen peroxide and decreases in beta-galactosidase activity were observed when compared to cells incubated without hydrogen peroxide. When cells, which expressed the intact GAL4 binding protein, were cultured in the presence of hydrogen peroxide the opposite was observed. Incubation of cells with buthionine sulfoximine augmented these responses to hydrogen peroxide. These data suggest that one of the mechanisms by which oxidative stress acts is via the modulation of protein-protein interactions and demonstrate that the yeast two-hybrid system may be a model by which to study protein interactions due to oxidative stress.     

Antioxidative protection in the central nervous ganglia of a mollusc Lymnaea stagnalis at modulation of activity of the NO-ergic system

In experiments on molluscs Lymnnaea stagnalis the state of antioxidative protection is studied in central nervous ganglia during a long-term activation (inhibition) of synthesis of nitric monoxide (NO) in the body. Effect of the blocker of NO-synthase N(G)-nitro-L-arginine (L-NNA) at the background of enhancement of pulmonary respiration has been established to be associated with a rise of levels of reduced glutathione and TBK-active products in the nervous tissue at preservation of a relatively high superoxide dismutase activity and a low glutathione peroxidase activity as compared with control group and the animals treated with the metabolic precursor of NO synthesis L-arginine. In spite of the revealed disturbances of balance of the body pro- and antioxidative system, DNA electrophoresis detected no products of its degradation, which can indicate the absence of massive programmed death of the nervous tissue cells in Lymnaea stagnalis during modulation of activity of the NO-ergic system.     

Hydrogen peroxide and hydroxyl radicals mediate palmitate-induced cytotoxicity to hepatoma cells: relation to mitochondrial permeability transition

We studied the toxicological responses of a human hepatoblastoma cell line (HepG2/C3A) to various free fatty acids (FFA) in order to identify the relation between reactive oxygen species (ROS) production and mitochondrial permeability transition (MPT). Exposure to the saturated FFA, palmitate, led to a time-dependent ROS production and hydrogen peroxide release as well as a loss of mitochondrial potential. The cytotoxicity of palmitate was significantly reduced by treating with scavengers of hydrogen peroxide, hydroxyl radical and the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butyl nitrone (POBN). Superoxide dismutase (SOD) mimics, nitric oxide scavenger, and inhibitor of de novo ceramide synthesis had no effect on the toxicity. MPT-inhibitor, cyclosporine, prevented the loss of mitochondrial potential but did not reduce the cytotoxicity. In contrast, inhibiting mitochondrial complexes I and III reduced the early potential loss and the cytotoxicity. These results suggest that palmitate-cytotoxicity to hepatoma cells is mediated through the production of H₂O₂ and *OH and independent of MPT.     

TORC1‐mediated sensing of chaperone activity alters glucose metabolism and extends lifespan

Protein quality control mechanisms, required for normal cellular functioning, encompass multiple functions related to protein production and maintenance. However, the existence of communication between proteostasis and metabolic networks and its underlying mechanisms remain elusive. Here, we report that enhanced chaperone activity and consequent improved proteostasis are sensed by TORC1 via the activity of Hsp82. Chaperone enrichment decreases the level of Hsp82, which deactivates TORC1 and leads to activation of Snf1/AMPK, regardless of glucose availability. This mechanism culminates in the extension of yeast replicative lifespan (RLS) that is fully reliant on both TORC1 deactivation and Snf1/AMPK activation. Specifically, we identify oxygen consumption increase as the downstream effect of Snf1 activation responsible for the entire RLS extension. Our results set a novel paradigm for the role of proteostasis in aging: modulation of the misfolded protein level can affect cellular metabolic features as well as mitochondrial activity and consequently modify lifespan. The described mechanism is expected to open new avenues for research of aging and age‐related diseases.     

Respiratory and TCA cycle activities affect <Emphasis Type="Italic">S. cerevisiae</Emphasis> lifespan,response to caloric restriction and mtDNA stability

We studied the importance of respiratory fitness in S. cerevisiae lifespan, response to caloric restriction (CR) and mtDNA stability. Mutants harboring mtDNA instability and electron transport defects do not respond to CR, while tricarboxylic acid cycle mutants presented extended lifespans due to CR. Interestingly, mtDNA is unstable in cells lacking dihydrolipoyl dehydrogenase under CR conditions, and cells lacking aconitase under standard conditions (both enzymes are components of the TCA and mitochondrial nucleoid). Altogether, our data indicate that respiratory integrity is required for lifespan extension by CR and that mtDNA stability is regulated by nucleoid proteins in a glucose-sensitive manner.     

NQR1 controls lifespan by regulating the promotion of respiratory metabolism in yeast

The activity and expression of plasma membrane NADH coenzyme Q reductase is increased by calorie restriction (CR) in rodents. Although this effect is well-established and is necessary for CR's ability to delay aging, the mechanism is unknown. Here we show that the Saccharomyces cerevisiae homolog, NADH-Coenzyme Q reductase 1 (NQR1), resides at the plasma membrane and when overexpressed extends both replicative and chronological lifespan. We show that NQR1 extends replicative lifespan in a SIR2-dependent manner by shifting cells towards respiratory metabolism. Chronological lifespan extension, in contrast, occurs via an SIR2-independent decrease in ethanol production. We conclude that NQR1 is a key mediator of lifespan extension by CR through its effects on yeast metabolism and discuss how these findings could suggest a function for this protein in lifespan extension in mammals.     

The Fission Yeast php2 Mutant Displays a Lengthened Chronological Lifespan

The Schizosaccharomyces pombe php2 ⁺ gene encodes a subunit of the CCAAT-binding factor complex. We found that disruption of the php2 ⁺ gene extended the chronological lifespan of the fission yeast. Moreover, the lifespan of the Δphp2 mutant was barely extended under calorie restricted (CR) conditions. Many other phenotypes of the Δphp2 mutant resembled those of wild-type cells grown under CR conditions, suggesting that the Δphp2 mutant might undergo CR. The mutant also showed low respiratory activity concomitant with decreased expression of the cyc1 ⁺ and rip1 ⁺ genes, both of which are involved in mitochondrial electron transport. On the basis of a chromatin immunoprecipitation assay, we determined that Php2 binds to a DNA region upstream of cyc1 ⁺ and rip1 ⁺ in S. pombe. Here we discuss the possible mechanisms by which the chronological lifespan of Δphp2 mutant is extended.     

Unopposed mitochondrial fission leads to severe lifespan shortening

Mitochondrial morphology is controlled by the opposing processes of fusion and fission. Previously, in baker’s yeast it was shown that reduced mitochondrial fission leads to a network-like morphology, decreased sensitivity for the induction of apoptosis and a remarkable extension of both replicative and chronological lifespan. However, the effects of reduced mitochondrial fusion on aging are so far unknown and complicated by the fact that deletion of genes encoding components of mitochondrial fusion are often lethal to higher organisms. This is also true for the mammalian OPA1 protein, which is a key regulator of mitochondrial inner membrane fusion. Baker’s yeast contains an OPA1 ortholog, Mgm1p. Deletion of Mgm1 is possible in yeast due to the fact that mitochondrial function is not essential for growth on glucose-containing media. In this study, we report that absence of mitochondrial fusion in the Δmgm1 mutant leads to a striking reduction of both replicative and chronological lifespan. Concomitantly, sensitivity to apoptosis elicitation via the reactive oxygen species hydrogen peroxide is substantially increased. These results demonstrate that the unopposed mitochondrial fission as displayed by the Δmgm1 mutant strongly affects organismal aging. Moreover, our results bear important clues for translational research to intervene into age-related degenerative processes also in multicellular organisms including humans.