Doreen Kuhlow

Role of Sirtuins in Lifespan Regulation is Linked to Methylation of Nicotinamide

Sirtuins, a family of histone deacetylases, have a fiercely debated role in regulating lifespan. In contrast with recent observations, here we find that overexpression of sir-2.1, the ortholog of mammalian SirT1, does extend Caenorhabditis elegans lifespan. Sirtuins mandatorily convert NAD(+) into nicotinamide (NAM). We here find that NAM and its metabolite, 1-methylnicotinamide (MNA), extend C. elegans lifespan, even in the absence of sir-2.1. We identify a previously unknown C. elegans nicotinamide-N-methyltransferase, encoded by a gene now named anmt-1, to generate MNA from NAM. Disruption and overexpression of anmt-1 have opposing effects on lifespan independent of sirtuins, with loss of anmt-1 fully inhibiting sir-2.1-mediated lifespan extension. MNA serves as a substrate for a newly identified aldehyde oxidase, GAD-3, to generate hydrogen peroxide, which acts as a mitohormetic reactive oxygen species signal to promote C. elegans longevity. Taken together, sirtuin-mediated lifespan extension depends on methylation of NAM, providing an unexpected mechanistic role for sirtuins beyond histone deacetylation.

Mitochondrial hormesis links low-dose arsenite exposure to lifespan extension.

Arsenite is one of the most toxic chemical substances known and is assumed to exert detrimental effects on viability even at lowest concentrations. By contrast and unlike higher concentrations, we here find that exposure to low‐dose arsenite promotes growth of cultured mammalian cells. In the nematode C. elegans, low‐dose arsenite promotes resistance against thermal and chemical stressors and extends lifespan of this metazoan, whereas higher concentrations reduce longevity. While arsenite causes a transient increase in reactive oxygen species (ROS) levels in C. elegans, co‐exposure to ROS scavengers prevents the lifespan‐extending capabilities of arsenite, indicating that transiently increased ROS levels act as transducers of arsenite effects on lifespan, a process known as mitohormesis. This requires two transcription factors, namely DAF‐16 and SKN‐1, which employ the metallothionein MTL‐2 as well as the mitochondrial transporter TIN‐9.1 to extend lifespan. Taken together, low‐dose arsenite extends lifespan, providing evidence for nonlinear dose‐response characteristics of toxin‐mediated stress resistance and longevity in a multicellular organism.

D -Glucosamine supplementation extends life span of nematodes and of ageing mice

D-Glucosamine (GlcN) is a freely available and commonly used dietary supplement potentially promoting cartilage health in humans, which also acts as an inhibitor of glycolysis. Here we show that GlcN, independent of the hexosamine pathway, extends Caenorhabditis elegans life span by impairing glucose metabolism that activates AMP-activated protein kinase (AMPK/AAK-2) and increases mitochondrial biogenesis. Consistent with the concept of mitohormesis, GlcN promotes increased formation of mitochondrial reactive oxygen species (ROS) culminating in increased expression of the nematodal amino acid-transporter 1 (aat-1) gene. Ameliorating mitochondrial ROS formation or impairment of aat-1-expression abolishes GlcN-mediated life span extension in an NRF2/SKN-1-dependent fashion. Unlike other calorie restriction mimetics, such as 2-deoxyglucose, GlcN extends life span of ageing C57BL/6 mice, which show an induction of mitochondrial biogenesis, lowered blood glucose levels, enhanced expression of several murine amino-acid transporters, as well as increased amino-acid catabolism. Taken together, we provide evidence that GlcN extends life span in evolutionary distinct species by mimicking a low-carbohydrate diet.

Branched-chain amino acid catabolism is a conserved regulator of physiological ageing

Ageing has been defined as a global decline in physiological function depending on both environmental and genetic factors. Here we identify gene transcripts that are similarly regulated during physiological ageing in nematodes, zebrafish and mice. We observe the strongest extension of lifespan when impairing expression of the branched-chain amino acid transferase-1 (bcat-1) gene in C. elegans, which leads to excessive levels of branched-chain amino acids (BCAAs). We further show that BCAAs reduce a LET-363/mTOR-dependent neuro-endocrine signal, which we identify as DAF-7/TGFβ, and that impacts lifespan depending on its related receptors, DAF-1 and DAF-4, as well as ultimately on DAF-16/FoxO and HSF-1 in a cell-non-autonomous manner. The transcription factor HLH-15 controls and epistatically synergizes with BCAT-1 to modulate physiological ageing. Lastly and consistent with previous findings in rodents, nutritional supplementation of BCAAs extends nematodal lifespan. Taken together, BCAAs act as periphery-derived metabokines that induce a central neuro-endocrine response, culminating in extended healthspan.

Variable Expression of Cre Recombinase Transgenes Precludes Reliable Prediction of Tissue-Specific Gene Disruption by Tail-Biopsy Genotyping

The Cre/loxP-system has become the system of choice for the generation of conditional so-called knockout mouse strains, i.e. the tissue-specific disruption of expression of a certain target gene. We here report the loss of expression of Cre recombinase in a transgenic mouse strain with increasing number of generations. This eventually led to the complete abrogation of gene expression of the inserted Cre cDNA while still being detectable at the genomic level. Conversely, loss of Cre expression caused an incomplete or even complete lack of disruption for the protein under investigation. As Cre expression in the tissue of interest in most cases cannot be addressed in vivo during the course of a study, our findings implicate the possibility that individual tail-biopsy genotypes may not necessarily indicate the presence or absence of gene disruption. This indicates that sustained post hoc analyses in regards to efficacy of disruption for every single study group member may be required.

The Friedreich's ataxia protein frataxin modulates DNA base excision repair in prokaryotes and mammals

DNA-repair mechanisms enable cells to maintain their genetic information by protecting it from mutations that may cause malignant growth. Recent evidence suggests that specific DNA-repair enzymes contain ISCs (iron–sulfur clusters). The nuclearencoded protein frataxin is essential for the mitochondrial biosynthesis of ISCs. Frataxin deficiency causes a neurodegenerative disorder named Friedreichs ataxia in humans. Various types of cancer occurring at young age are associated with this disease, and hence with frataxin deficiency. Mice carrying a hepatocyte-specific disruption of the frataxin gene develop multiple liver tumours for unresolved reasons. In the present study, we show that frataxin deficiency in murine liver is associated with increased basal levels of oxidative DNA base damage. Accordingly, eukaryotic V79 fibroblasts overexpressing human frataxin show decreased basal levels of these modifications, while prokaryotic Salmonella enterica serotype Typhimurium TA104 strains transformed with human frataxin show decreased mutation rates. The repair rates of oxidative DNA base modifications in V79 cells overexpressing frataxin were significantly higher than in control cells. Lastly, cleavage activity related to the ISC-independent repair enzyme 8-oxoguanine glycosylase was found to be unaltered by frataxin overexpression. These findings indicate that frataxin modulates DNA-repair mechanisms probably due to its impact on ISC-dependent repair proteins, linking mitochondrial dysfunction to DNA repair and tumour initiation.

Pharmacological and Genetic Manipulation of p53 in Brown Fat at Adult But Not Embryonic Stages Regulates Thermogenesis and Body Weight in Male Mice

p53 is a well-known tumor suppressor that plays multiple biological roles, including the capacity to modulate metabolism at different levels. However, its metabolic role in brown adipose tissue (BAT) remains largely unknown. Herein we sought to investigate the physiological role of endogenous p53 in BAT and its implication on BAT thermogenic activity and energy balance. To this end, we generated and characterized global p53-null mice and mice lacking p53 specifically in BAT. Additionally we performed gain-and-loss-of-function experiments in the BAT of adult mice using virogenetic and pharmacological approaches. BAT was collected and analyzed by immunohistochemistry, thermography, real-time PCR, and Western blot. p53-deficient mice were resistant to diet-induced obesity due to increased energy expenditure and BAT activity. However, the deletion of p53 in BAT using a Myf5-Cre driven p53 knockout did not show any changes in body weight or the expression of thermogenic markers. The acute inhibition of p53 in the BAT of adult mice slightly increased body weight and inhibited BAT thermogenesis, whereas its overexpression in the BAT of diet-induced obese mice reduced body weight and increased thermogenesis. On the other hand, pharmacological activation of p53 improves body weight gain due to increased BAT thermogenesis by sympathetic nervous system in obese adult wild-type mice but not in p53(-/-) animals. These results reveal that p53 regulates BAT metabolism by coordinating body weight and thermogenesis, but these metabolic actions are tissue specific and also dependent on the developmental stage.

Opposing effects of dietary sugar and saturated fat on cardiovascular risk factors and glucose metabolism in mitochondrially impaired mice.

PurposeBoth dietary fat and dietary sucrose are major components of Western diets that may differentially affect the risk for body mass gain, diabetes mellitus, and cardiovascular disease.MethodsWe have phenotypically analyzed mice with ubiquitously impaired expression of mitochondrial frataxin protein that were challenged with diets differing in macronutrient content, namely high-sucrose/low-fat and high-saturated fat/low-sugar diets.ResultsWe find here that a high-sucrose/low-fat diet has especially detrimental effects in mice with impaired mitochondrial metabolism promoting several independent cardiovascular risk factors, including impaired glucose metabolism, fasting hyperinsulinemia, reduced glucose-stimulated insulin secretion, increased serum triglycerides, and elevated cholesterol levels due to increased expression of HMG-CoA reductase. In contrast, a high-saturated fat/low-sugar diet protects mice with impaired mitochondrial metabolism from diet-induced obesity by increasing total energy expenditure and increasing expression of ACAA2, a rate-limiting enzyme of mitochondrial beta-oxidation, whereas no concomitant improvement of glucose metabolism was observed.ConclusionsTaken together, our results suggest that mitochondrial dysfunction may cause sucrose to become a multifunctional cardiovascular risk factor, whereas low-sugar diets high in saturated fat may prevent weight gain without improving glucose metabolism.

Impairment of insulin signalling in peripheral tissue fails to extend murine lifespan

Impaired insulin/IGF1 signalling has been shown to extend lifespan in model organisms ranging from yeast to mammals. Here we sought to determine the effect of targeted disruption of the insulin receptor (IR) in non‐neuronal tissues of adult mice on the lifespan. We induced hemizygous (PerIRKO+/−) or homozygous (PerIRKO−/−) disruption of the IR in peripheral tissue of 15‐weeks‐old mice using a tamoxifen‐inducible Cre transgenic mouse with only peripheral tissue expression, and subsequently monitored glucose metabolism, insulin signalling and spontaneous death rates over 4 years. Complete peripheral IR disruption resulted in a diabetic phenotype with increased blood glucose and plasma insulin levels in young mice. Although blood glucose levels returned to normal, and fat mass was reduced in aged PerIRKO−/− mice, their lifespan was reduced. By contrast, heterozygous disruption had no effect on lifespan. This was despite young male PerIRKO+/− mice showing reduced fat mass and mild increase in hepatic insulin sensitivity. In conflict with findings in metazoans like Caenorhabditis elegans and Drosophila melanogaster, our results suggest that heterozygous impairment of the insulin signalling limited to peripheral tissues of adult mice fails to extend lifespan despite increased systemic insulin sensitivity, while homozygous impairment shortens lifespan.