Hepatic hydrogen sulfide levels are reduced in mouse model of Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare human disease characterised by accelerated biological ageing. Current treatments are limited, and most patients die before 15 years of age. Hydrogen sulfide (H2S) is an important gaseous signalling molecule that it central to multiple cellular homeostasis mechanisms. Dysregulation of tissue H2S levels is thought to contribute to an ageing phenotype in many tissues across animal models. Whether H2S is altered in HGPS is unknown. We investigated hepatic H2S production capacity and transcript, protein and enzymatic activity of proteins that regulate hepatic H2S production and disposal in a mouse model of HGPS (G609G mice, mutated Lmna gene equivalent to a causative mutation in HGPS patients). G609G mice were maintained on either regular chow (RC) or high fat diet (HFD), as HFD has been previously shown to significantly extend lifespan of G609G mice, and compared to wild type (WT) mice maintained on RC. RC fed G609G mice had significantly reduced hepatic H2S production capacity relative to WT mice, with a compensatory elevation in mRNA transcripts associated with several H2S production enzymes, including cystathionine-γ-lyase (CSE). H2S levels and CSE protein were partially rescued in HFD fed G609G mice. As current treatments for patients with HGPS have failed to confer significant improvements to symptoms or longevity, the need for novel therapeutic targets is acute and the regulation of H2S through dietary or pharmacological means may be a promising new avenue for research.

RNA Polymerase III, Ageing and Longevity.

Transcription in eukaryotic cells is performed by three RNA polymerases. RNA polymerase I synthesises most rRNAs, whilst RNA polymerase II transcribes all mRNAs and many non-coding RNAs. The largest of the three polymerases is RNA polymerase III (Pol III) which transcribes a variety of short non-coding RNAs including tRNAs and the 5S rRNA, in addition to other small RNAs such as snRNAs, snoRNAs, SINEs, 7SL RNA, Y RNA, and U6 spilceosomal RNA. Pol III-mediated transcription is highly dynamic and regulated in response to changes in cell growth, cell proliferation and stress. Pol III-generated transcripts are involved in a wide variety of cellular processes, including translation, genome and transcriptome regulation and RNA processing, with Pol III dys-regulation implicated in diseases including leukodystrophy, Alzheimer's, Fragile X-syndrome and various cancers. More recently, Pol III was identified as an evolutionarily conserved determinant of organismal lifespan acting downstream of mTORC1. Pol III inhibition extends lifespan in yeast, worms and flies, and in worms and flies acts from the intestine and intestinal stem cells respectively to achieve this. Intriguingly, Pol III activation achieved through impairment of its master repressor, Maf1, has also been shown to promote longevity in model organisms, including mice. In this review we introduce the Pol III transcription apparatus and review the current understanding of RNA Pol III's role in ageing and lifespan in different model organisms. We then discuss the potential of Pol III as a therapeutic target to improve age-related health in humans.

Differences in mitochondrial efficiency explain individual variation in growth performance.

The physiological causes of intraspecific differences in fitness components such as growth rate are currently a source of debate. It has been suggested that differences in energy metabolism may drive variation in growth, but it remains unclear whether covariation between growth rates and energy metabolism is: (i) a result of certain individuals acquiring and consequently allocating more resources to growth, and/or is (ii) determined by variation in the efficiency with which those resources are transformed into growth. Studies of individually housed animals under standardized nutritional conditions can help shed light on this debate. Here we quantify individual variation in metabolic efficiency in terms of the amount of adenosine triphosphate (ATP) generated per molecule of oxygen consumed by liver and muscle mitochondria and examine its effects, both on the rate of protein synthesis within these tissues and on the rate of whole-body growth of individually fed juvenile brown trout (Salmo trutta) receiving either a high or low food ration. As expected, fish on the high ration on average gained more in body mass and protein content than those maintained on the low ration. Yet, growth performance varied more than 10-fold among individuals on the same ration, resulting in some fish on low rations growing faster than others on the high ration. This variation in growth for a given ration was related to individual differences in mitochondrial properties: a high whole-body growth performance was associated with high mitochondrial efficiency of ATP production in the liver. Our results show for the first time, to our knowledge, that among-individual variation in the efficiency with which substrates are converted into ATP can help explain marked variation in growth performance, independent of food intake. This study highlights the existence of inter-individual differences in mitochondrial efficiency and its potential importance in explaining intraspecific variation in whole-animal performance.

The RCR and ATP/O Indices Can Give Contradictory Messages about Mitochondrial Efficiency.

Mitochondrial efficiency is typically taken to represent an animal's capacity to convert its resources into ATP. However, the term mitochondrial efficiency, as currently used in the literature, can be calculated as either the respiratory control ratio, RCR (ratio of mitochondrial respiration supporting ATP synthesis to that required to offset the proton leak) or as the amount of ATP generated per unit of oxygen consumed, ATP/O ratio. The question of how flexibility in mitochondrial energy properties (i.e., in rates of respiration to support ATP synthesis and offset proton leak, and in the rate of ATP synthesis) affects these indices of mitochondrial efficiency has tended to be overlooked. Furthermore, little is known of whether the RCR and ATP/O ratio vary in parallel, either among individuals or in response to environmental conditions. Using data from brown trout Salmo trutta we show that experimental conditions affect mitochondrial efficiency, but the apparent direction of change depends on the index chosen: a reduction in food availability was associated with an increased RCR (i.e., increased efficiency) but a decreased ATP/O ratio (decreased efficiency) in liver mitochondria. Moreover, there was a negative correlation across individuals held in identical conditions between their RCR and their ATP/O ratio. These results show that the choice of index of mitochondrial efficiency can produce different, even opposing, conclusions about the capacity of the mitochondria to produce ATP. Neither ratio is necessarily a complete measure of efficiency of ATP production in the living animal (RCR because it contains no assessment of ATP production, and ATP/O because it contains no assessment of respiration to offset the proton leak). Consequently, we suggest that a measure of mitochondrial efficiency obtained nearer to conditions where respiration simultaneously offsets the proton leak and produce ATP would be sensitive to changes in both proton leakage and ATP production, and is thus likely to be more representative of the state of the mitochondria in vivo.

Chronic helminth infection burden differentially affects haematopoietic cell development while ageing selectively impairs adaptive responses to infection.

Throughout the lifespan of an individual, the immune system undergoes complex changes while facing novel and chronic infections. Helminths, which infect over one billion people and impose heavy livestock productivity losses, typically cause chronic infections by avoiding and suppressing host immunity. Yet, how age affects immune responses to lifelong parasitic infection is poorly understood. To disentangle the processes involved, we employed supervised statistical learning techniques to identify which factors among haematopoietic stem and progenitor cells (HSPC), and both innate and adaptive responses regulate parasite burdens and how they are affected by host age. Older mice harboured greater numbers of the parasites' offspring than younger mice. Protective immune responses that did not vary with age were dominated by HSPC, while ageing specifically eroded adaptive immunity, with reduced numbers of naïve T cells, poor T cell responsiveness to parasites, and impaired antibody production. We identified immune factors consistent with previously-reported immune responses to helminths, and also revealed novel interactions between helminths and HSPC maturation. Our approach thus allowed disentangling the concurrent effects of ageing and infection across the full maturation cycle of the immune response and highlights the potential of such approaches to improve understanding of the immune system within the whole organism.

Hypothalamic-Pituitary Axis Regulates Hydrogen Sulfide Production.

Decreased growth hormone (GH) and thyroid hormone (TH) signaling are associated with longevity and metabolic fitness. The mechanisms underlying these benefits are poorly understood, but may overlap with those of dietary restriction (DR), which imparts similar benefits. Recently we discovered that hydrogen sulfide (H2S) is increased upon DR and plays an essential role in mediating DR benefits across evolutionary boundaries. Here we found increased hepatic H2S production in long-lived mouse strains of reduced GH and/or TH action, and in a cell-autonomous manner upon serum withdrawal in vitro. Negative regulation of hepatic H2S production by GH and TH was additive and occurred via distinct mechanisms, namely direct transcriptional repression of the H2S-producing enzyme cystathionine γ-lyase (CGL) by TH, and substrate-level control of H2S production by GH. Mice lacking CGL failed to downregulate systemic T4 metabolism and circulating IGF-1, revealing an essential role for H2S in the regulation of key longevity-associated hormones.

Simultaneous measurement of mitochondrial respiration and ATP production in tissue homogenates and calculation of effective P/O ratios.

The use of tissue homogenate has greatly aided the study of the functioning of mitochondria. However, the amount of ATP produced per oxygen molecule consumed, that is, the effective P/O ratio, has never been measured directly in tissue homogenate. Here we combine and refine existing methods previously used in permeabilized cells and isolated mitochondria to simultaneously measure mitochondrial ATP production (JATP) and oxygen consumption (JO2) in tissue homogenate. A major improvement over existing methods is in the control of ATPases that otherwise interfere with the ATP assay: our modified technique facilitates simultaneous measurement of the rates of "uncorrected" ATP synthesis and of ATP hydrolysis, thus minimizing the amount of tissue and time needed. Finally, we develop a novel method of calculating effective P/O ratios which corrects measurements of JATP and JO2 for rates of nonmitochondrial ATP hydrolysis and respiration, respectively. Measurements of JATP and JO2 in liver homogenates from brown trout (Salmo trutta) were highly reproducible, although activity declined once homogenates were 2 h old. We compared mitochondrial properties from fed and food-deprived animals to demonstrate that the method can detect mitochondrial flexibility in P/O ratios in response to nutritional state. This method simplifies studies examining the mitochondrial bioenergetics of tissue homogenates, obviating the need for differential centrifugation or chemical permeabilization and avoiding the use of nonmitochondrial ATPase inhibitors. We conclude that our approach for characterizing effective P/O ratio opens up new possibilities in the study of mitochondrial function in very small samples, where the use of other methods is limited.

Evidence that hematopoietic stem cell function is preserved during aging in long-lived S6K1 mutant mice.

The mechanistic target of rapamycin (mTOR) signalling pathway plays a highly conserved role in aging; mice lacking ribosomal protein S6 kinase 1 (S6K1-/-) have extended lifespan and healthspan relative to wild type (WT) controls. Exactly how reduced mTOR signalling induces such effects is unclear, although preservation of stem cell function may be important. We show, using gene expression analyses, that there was a reduction in expression of cell cycle genes in young (12 week) and aged (80 week) S6K1-/- BM-derived c-Kit+ cells when compared to age-matched WT mice, suggesting that these cells are more quiescent in S6K1-/- mice. In addition, we investigated hematopoietic stem cell (HSC) frequency and function in young and aged S6K1-/-and WT mice. Young, but not aged, S6K1-/-mice had more LSK (lineage-, c-Kit+, Sca-1+) cells (% of bone marrow (BM)), including the most primitive long-term repopulating HSCs (LT-HSC) relative to WT controls. Donor-derived engraftment of LT-HSCs in recipient mice was unaffected by genotype in young mice, but was enhanced in transplants using LT-HSCs derived from aged S6K1-/- mice. Our results are the first to provide evidence that age-associated HSC functional decline is ameliorated in a long-lived mTOR mutant mouse.

Variation in the link between oxygen consumption and ATP production, and its relevance for animal performance.

It is often assumed that an animal's metabolic rate can be estimated through measuring the whole-organism oxygen consumption rate. However, oxygen consumption alone is unlikely to be a sufficient marker of energy metabolism in many situations. This is due to the inherent variability in the link between oxidation and phosphorylation; that is, the amount of adenosine triphosphate (ATP) generated per molecule of oxygen consumed by mitochondria (P/O ratio). In this article, we describe how the P/O ratio can vary within and among individuals, and in response to a number of environmental parameters, including diet and temperature. As the P/O ratio affects the efficiency of cellular energy production, its variability may have significant consequences for animal performance, such as growth rate and reproductive output. We explore the adaptive significance of such variability and hypothesize that while a reduction in the P/O ratio is energetically costly, it may be associated with advantages in terms of somatic maintenance through reduced production of reactive oxygen species. Finally, we discuss how considering variation in mitochondrial efficiency, together with whole-organism oxygen consumption, can permit a better understanding of the relationship between energy metabolism and life history for studies in evolutionary ecology.

Longevity and skeletal muscle mass: the role of IGF signalling, the sirtuins, dietary restriction and protein intake.

Advancing age is associated with a progressive loss of skeletal muscle (SkM) mass and function. Given the worldwide aging demographics, this is a major contributor to morbidity, escalating socio-economic costs and ultimately mortality. Previously, it has been established that a decrease in regenerative capacity in addition to SkM loss with age coincides with suppression of insulin/insulin-like growth factor signalling pathways. However, genetic or pharmacological modulations of these highly conserved pathways have been observed to significantly enhance life and healthspan in various species, including mammals. This therefore provides a controversial paradigm in which reduced regenerative capacity of skeletal muscle tissue with age potentially promotes longevity of the organism. This paradox will be assessed and considered in the light of the following: (i) the genetic knockout, overexpression and pharmacological models that induce lifespan extension (e.g. IRS-1/s6K KO, mTOR inhibition) versus the important role of these signalling pathways in SkM growth and adaptation; (ii) the role of the sirtuins (SIRTs) in longevity versus their emerging role in SkM regeneration and survival under catabolic stress; (iii) the role of dietary restriction and its impact on longevity versus skeletal muscle mass regulation; (iv) the crosstalk between cellular energy metabolism (AMPK/TSC2/SIRT1) and survival (FOXO) versus growth and repair of SkM (e.g. AMPK vs. mTOR); and (v) the impact of protein feeding in combination with dietary restriction will be discussed as a potential intervention to maintain SkM mass while increasing longevity and enabling healthy aging.

Testing the Effects of DL-Alpha-Tocopherol Supplementation on Oxidative Damage, Total Antioxidant Protection and the Sex-Specific Responses of Reproductive Effort and Lifespan to Dietary Manipulation in Australian Field Crickets (Teleogryllus commodus).

The oxidative stress theory predicts that the accumulation of oxidative damage causes aging. More generally, oxidative damage could be a cost of reproduction that reduces survival. Both of these hypotheses have mixed empirical support. To better understand the life-history consequences of oxidative damage, we fed male and female Australian field crickets (Teleogryllus commodus) four diets differing in their protein and carbohydrate content, which have sex-specific effects on reproductive effort and lifespan. We supplemented half of these crickets with the vitamin E isoform DL-alpha-tocopherol and measured the effects of nutrient intake on lifespan, reproduction, oxidative damage and antioxidant protection. We found a clear trade-off between reproductive effort and lifespan in females but not in males. In direct contrast to the oxidative stress theory, crickets fed diets that improved their lifespan had high levels of oxidative damage to proteins. Supplementation with DL-alpha-tocopherol did not significantly improve lifespan or reproductive effort. However, males fed diets that increased their reproductive investment experienced high oxidative damage to proteins. While this suggests that male reproductive effort could elevate oxidative damage, this was not associated with reduced male survival. Overall, these results provide little evidence that oxidative damage plays a central role in mediating life-history trade-offs in T. commodus.

Lifespan modulation in mice and the confounding effects of genetic background.

We are currently in the midst of a revolution in ageing research, with several dietary, genetic and pharmacological interventions now known to modulate ageing in model organisms. Excitingly, these interventions also appear to have beneficial effects on late-life health. For example, dietary restriction (DR) has been shown to slow the incidence of age-associated cardiovascular disease, metabolic disease, cancer and brain ageing in non-human primates and has been shown to improve a range of health indices in humans. While the idea that DR's ability to extend lifespan is often thought of as being universal, studies in a range of organisms, including yeast, mice and monkeys, suggest that this may not actually be the case. The precise reasons underlying these differential effects of DR on lifespan are currently unclear, but genetic background may be an important factor in how an individual responds to DR. Similarly, recent findings also suggest that the responsiveness of mice to specific genetic or pharmacological interventions that modulate ageing may again be influenced by genetic background. Consequently, while there is a clear driver to develop interventions to improve late-life health and vitality, understanding precisely how these act in response to particular genotypes is critical if we are to translate these findings to humans. We will consider of the role of genetic background in the efficacy of various lifespan interventions and discuss potential routes of utilising genetic heterogeneity to further understand how particular interventions modulate lifespan and healthspan.

Deleterious consequences of antioxidant supplementation on lifespan in a wild-derived mammal.

While oxidative damage owing to reactive oxygen species (ROS) often increases with advancing age and is associated with many age-related diseases, its causative role in ageing is controversial. In particular, studies that have attempted to modulate ROS-induced damage, either upwards or downwards, using antioxidant or genetic approaches, generally do not show a predictable effect on lifespan. Here, we investigated whether dietary supplementation with either vitamin E (α-tocopherol) or vitamin C (ascorbic acid) affected oxidative damage and lifespan in short-tailed field voles, Microtus agrestis. We predicted that antioxidant supplementation would reduce ROS-induced oxidative damage and increase lifespan relative to unsupplemented controls. Antioxidant supplementation for nine months reduced hepatic lipid peroxidation, but DNA oxidative damage to hepatocytes and lymphocytes was unaffected. Surprisingly, antioxidant supplementation significantly shortened lifespan in voles maintained under both cold (7 ± 2°C) and warm (22 ± 2°C) conditions. These data further question the predictions of free-radical theory of ageing and critically, given our previous research in mice, indicate that similar levels of antioxidants can induce widely different interspecific effects on lifespan.

Mammalian models of extended healthy lifespan.

Over the last two centuries, there has been a significant increase in average lifespan expectancy in the developed world. One unambiguous clinical implication of getting older is the risk of experiencing age-related diseases including various cancers, dementia, type-2 diabetes, cataracts and osteoporosis. Historically, the ageing process and its consequences were thought to be intractable. However, over the last two decades or so, a wealth of empirical data has been generated which demonstrates that longevity in model organisms can be extended through the manipulation of individual genes. In particular, many pathological conditions associated with the ageing process in model organisms, and importantly conserved from nematodes to humans, are attenuated in long-lived genetic mutants. For example, several long-lived genetic mouse models show attenuation in age-related cognitive decline, adiposity, cancer and glucose intolerance. Therefore, these long-lived mice enjoy a longer period without suffering the various sequelae of ageing. The greatest challenge in the biology of ageing is to now identify the mechanisms underlying increased healthy lifespan in these model organisms. Given that the elderly are making up an increasingly greater proportion of society, this focused approach in model organisms should help identify tractable interventions that can ultimately be translated to humans.

Vitamin E supplementation and mammalian lifespan.

Vitamin E refers to a family of several compounds that possess a similar chemical structure comprising a chromanol ring with a 16-carbon side chain. The degree of saturation of the side chain, and positions and nature of methyl groups designate the compounds as tocopherols or tocotrienols. Vitamin E compounds have antioxidant properties due to a hydroxyl group on the chromanol ring. Recently, it has been suggested that vitamin E may also regulate signal transduction and gene expression. We previously reported that lifelong dietary vitamin E (alpha-tocopherol) supplementation significantly increased median lifespan in C57BL/6 mice by 15%. This lifespan extension appeared to be independent of any antioxidant effect. Employing a transcriptional approach, we suggest that this increase in lifespan may reflect an anti-cancer effect via induction of the P21 signalling pathway, since cancer is the major cause of death in small rodents. We suggest that the role of this pathway in life span extension following supplementation of vitamin E now requires further investigation.

Ribosomal protein S6 kinase 1 signaling regulates mammalian life span.

Caloric restriction (CR) protects against aging and disease, but the mechanisms by which this affects mammalian life span are unclear. We show in mice that deletion of ribosomal S6 protein kinase 1 (S6K1), a component of the nutrient-responsive mTOR (mammalian target of rapamycin) signaling pathway, led to increased life span and resistance to age-related pathologies, such as bone, immune, and motor dysfunction and loss of insulin sensitivity. Deletion of S6K1 induced gene expression patterns similar to those seen in CR or with pharmacological activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK), a conserved regulator of the metabolic response to CR. Our results demonstrate that S6K1 influences healthy mammalian life-span and suggest that therapeutic manipulation of S6K1 and AMPK might mimic CR and could provide broad protection against diseases of aging.

Deletion of the von Hippel-Lindau gene in pancreatic beta cells impairs glucose homeostasis in mice.

Defective insulin secretion in response to glucose is an important component of the beta cell dysfunction seen in type 2 diabetes. As mitochondrial oxidative phosphorylation plays a key role in glucose-stimulated insulin secretion (GSIS), oxygen-sensing pathways may modulate insulin release. The von Hippel-Lindau (VHL) protein controls the degradation of hypoxia-inducible factor (HIF) to coordinate cellular and organismal responses to altered oxygenation. To determine the role of this pathway in controlling glucose-stimulated insulin release from pancreatic beta cells, we generated mice lacking Vhl in pancreatic beta cells (betaVhlKO mice) and mice lacking Vhl in the pancreas (PVhlKO mice). Both mouse strains developed glucose intolerance with impaired insulin secretion. Furthermore, deletion of Vhl in beta cells or the pancreas altered expression of genes involved in beta cell function, including those involved in glucose transport and glycolysis, and isolated betaVhlKO and PVhlKO islets displayed impaired glucose uptake and defective glucose metabolism. The abnormal glucose homeostasis was dependent on upregulation of Hif-1alpha expression, and deletion of Hif1a in Vhl-deficient beta cells restored GSIS. Consistent with this, expression of activated Hif-1alpha in a mouse beta cell line impaired GSIS. These data suggest that VHL/HIF oxygen-sensing mechanisms play a critical role in glucose homeostasis and that activation of this pathway in response to decreased islet oxygenation may contribute to beta cell dysfunction.

Life-long vitamin C supplementation in combination with cold exposure does not affect oxidative damage or lifespan in mice, but decreases expression of antioxidant protection genes.

Oxidative stress is suggested to be central to the ageing process, with endogenous antioxidant defence and repair mechanisms in place to minimize damage. Theoretically, supplementation with exogenous antioxidants might support the endogenous antioxidant system, thereby reducing oxidative damage, ageing-related functional decline and prolonging life- and health-span. Yet supplementation trials with antioxidants in animal models have had minimal success. Human epidemiological data are similarly unimpressive, leading some to question whether vitamin C, for example, might have pro-oxidant properties in vivo. We supplemented cold exposed (7+/-2 degrees C) female C57BL/6 mice over their lifespan with vitamin C (ascorbyl-2-polyphosphate), widely advocated and self administered to reduce oxidative stress, retard ageing and increase healthy lifespan. No effect on mean or maximum lifespan following vitamin C treatment or any significant impact on body mass, or on parameters of energy metabolism was observed. Moreover, no differences in hepatocyte and lymphocyte DNA oxidative damage or hepatic lipid peroxidation was seen between supplemented and control mice. Using a DNA macroarray specific for oxidative stress-related genes, we found that after 18 months of supplementation, mice exhibited a significantly reduced expression of several genes in the liver linked to free-radical scavenging, including Mn-superoxide dismutase. We confirmed these effects by Northern blotting and found additional down-regulation of glutathione peroxidase (not present on macroarray) in the vitamin C treated group. We suggest that high dietary doses of vitamin C are ineffective at prolonging lifespan in mice because any positive benefits derived as an antioxidant are offset by compensatory reductions in endogenous protection mechanisms, leading to no net reduction in accumulated oxidative damage.