Does Reduced IGF-1R Signaling in Igf1r+/− Mice Alter Aging?

Mutations in insulin/IGF-1 signaling pathway have been shown to lead to increased longevity in various invertebrate models. Therefore, the effect of the haplo- insufficiency of the IGF-1 receptor (Igf1r+/−) on longevity/aging was evaluated in C57Bl/6 mice using rigorous criteria where lifespan and end-of-life pathology were measured under optimal husbandry conditions using large sample sizes. Igf1r+/− mice exhibited reductions in IGF-1 receptor levels and the activation of Akt by IGF-1, with no compensatory increases in serum IGF-1 or tissue IGF-1 mRNA levels, indicating that the Igf1r+/− mice show reduced IGF-1 signaling. Aged male, but not female Igf1r+/− mice were glucose intolerant, and both genders developed insulin resistance as they aged. Female, but not male Igf1r+/− mice survived longer than wild type mice after lethal paraquat and diquat exposure, and female Igf1r+/− mice also exhibited less diquat-induced liver damage. However, no significant difference between the lifespans of the male Igf1r+/− and wild type mice was observed; and the mean lifespan of the Igf1r+/− females was increased only slightly (less than 5%) compared to wild type mice. A comprehensive pathological analysis showed no significant difference in end-of-life pathological lesions between the Igf1r+/− and wild type mice. These data show that the Igf1r+/− mouse is not a model of increased longevity and delayed aging as predicted by invertebrate models with mutations in the insulin/IGF-1 signaling pathway

Hepatic response to oxidative injury in long-lived Ames dwarf mice

Multiple stress resistance pathways were evaluated in the liver of Ames dwarf mice before and after exposure to the oxidative toxin diquat, seeking clues to the exceptional longevity conferred by this mutation. Before diquat treatment, Ames dwarf mice, compared with nonmutant littermate controls, had 2- to 6-fold higher levels of expression of mRNAs for immediate early genes and 2- to 5-fold higher levels of mRNAs for genes dependent on the transcription factor Nrf2. Diquat led to a 2-fold increase in phosphorylation of the stress kinase ERK in control (but not Ames dwarf) mice and to a 50% increase in phosphorylation of the kinase JNK2 in Ames dwarf (but not control) mice. Diquat induction of Nrf2 protein was higher in dwarf mice than in controls. Of 6 Nrf2-responsive genes evaluated, 4 (HMOX, NQO-1, MT-1, and MT-2) remained 2- to 10-fold lower in control than in dwarf liver after diquat, and the other 2 (GCLM and TXNRD) reached levels already seen in dwarf liver at baseline. Thus, livers of Ames dwarf mice differ systematically from controls in multiple stress resistance pathways before and after exposure to diquat, suggesting mechanisms for stress resistance and extended longevity in Ames dwarf mice.—Sun, L. Y., Bokov, A. F., Richardson, A., Miller, R. A. Hepatic response to oxidative injury in long-lived Ames dwarf mice

Profiling the Anaerobic Response of <i>C. elegans</i> Using GC-MS

<div><p>The nematode <i>Caenorhabditis elegans</i> is a model organism that has seen extensive use over the last four decades in multiple areas of investigation. In this study we explore the response of the nematode <i>Caenorhabditis elegans</i> to acute anoxia using gas-chromatography mass-spectrometry (GC-MS). We focus on the readily-accessible worm exometabolome to show that <i>C. elegans</i> are mixed acid fermenters that utilize several metabolic pathways in unconventional ways to remove reducing equivalents – including partial reversal of branched-chain amino acid catabolism and a potentially novel use of the glyoxylate pathway. In doing so, we provide detailed methods for the collection and analysis of excreted metabolites that, with minimal adjustment, should be applicable to many other species. We also describe a procedure for collecting highly volatile compounds from <i>C. elegans</i>. We are distributing our mass spectral library in an effort to facilitate wider use of metabolomics.</p></div

Assessment of technical and biological variability in samples.

<p>Replicate technical analyses were performed on each of two exometabolome samples collected from independent, normoxically-cultured, N2 worm preparations. For all four datasets, integrated metabolite peak areas were normalized to internal standard and total worm protein. Data were plotted after log₁₀ transformation. (<b>a</b>) Combined scatterplot showing technical variation for both biological samples (A1 versus A2, B1 versus B2). (<b>b</b>) Bland-Altman plot highlighting differences between the integrated intensities of metabolites in A1 and A2. The bold horizontal line represents the average difference across all metabolites. The solid horizontal lines correspond to the mean difference ± two standard deviations. The dashed vertical line is the experimentally-determined noise threshold. Data below this threshold were removed from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046140#pone-0046140-g003" target="_blank">Figure 3</a> by applying a low-abundance cutoff.</p

GC-MS reveals marked differences in the exometabolome of <i>C. elegans</i> cultured under anaerobic versus normoxic conditions.

<p>(<b>a</b>) Extracted peak areas for all metabolites were log transformed after normalization to an internal standard and total protein. Metabolites that differed significantly between conditions were identified using a mixed-modeling approach. A false discovery rate (FDR) of 5% was used to set the significance cutoffs. Shown are spaghetti plots for metabolites analyzed by the two different GC procedures described in Methods. Lines correspond to individual metabolites: red lines, significant increases under anoxia; blue lines, significant decreases; and gray lines, no significant change. To aid visual interpretation, the values plotted in this panel were scaled by being converted to z-scores. (<b>b</b> and <b>c</b>) Hierarchical clustering (based on Pearson's correlation coefficient) was used to segregate metabolites that were synchronously up- or down-regulated following oxygen removal (only significantly altered metabolites are plotted). A low-abundance cut-off filter was applied. Heat maps are colored according to: (i) individual metabolite variation across the sample set (blue-yellow); and (ii) global variation among metabolites over the entire exometabolome data set (blue-red). The latter method only approximates relative metabolite abundance (compare to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046140#pone.0046140.s001" target="_blank">Figure S1</a>). Exometabolites are plotted in two groups based on analytical separation technique, with volatile metabolites shown in (<b>b</b>) and remaining components in (<b>c</b>).</p

Technical Reproducibility.

<p>Technical Reproducibility.</p

Metabolic map illustrating intracellular changes predicted to occur based on the exometabolome of wild-type worms following exposure to 18 hours of anoxia.

<p>Major metabolic alterations characterize the <i>C. elegans</i> response to anoxia. Exometabolites that were identified by GC-MS, and which were used to drive map construction, are accompanied by bar graphs. In each graph, the incubation condition that resulted in the highest level of expression of a particular metabolite was assigned an expression value of one then the value in the other condition was scaled accordingly (<i>grey</i>: normoxic, <i>black</i>: anaerobic). Shown are the averages of five measurements, with error bars representing ± one standard deviation. Major flux pathways are color-coded as follows: <i>green</i>, glycolysis/malate dismutation/volatile fatty acid synthesis; <i>blue</i>, glyoxylate cycle; <i>pink</i>, acetate/ethanol fermentation; <i>yellow</i>, lactate fermentation/2-hydroxybutyrate fermentation. *, less than 10% false discovery rate; **, less than 5% false discovery rate; NADH, nicotinamide adenine dinucleotide-producing reactions; RQ, reduced rhodoquinone; O₂, oxygen consuming reactions; ^‡, methylacrylic acid was detected but resided on a shoulder of a background peak that we could not deconvolve fully. Circled numbers represent the following glycolysis by-pass enzymes of gluconeogenesis: 1, pyruvate carboxylase; 2, phosphoenolpyruvate carboxykinase; 3, fructose 1,6-bisphosphatase; 4, glucose-6-phosphatase. (Although not shown in the map, pyruvate is the source of acetyl CoA in the coupled conversion of succinate to succinyl CoA on the path to volatile fatty acid synthesis.)</p

DLK-1, SEK-3 and PMK-3 Are Required for the Life Extension Induced by Mitochondrial Bioenergetic Disruption in <i>C</i>. <i>elegans</i>

<div><p>Mitochondrial dysfunction underlies numerous age-related pathologies. In an effort to uncover how the detrimental effects of mitochondrial dysfunction might be alleviated, we examined how the nematode <i>C</i>. <i>elegans</i> not only adapts to disruption of the mitochondrial electron transport chain, but in many instances responds with extended lifespan. Studies have shown various retrograde responses are activated in these animals, including the well-studied ATFS-1-dependent mitochondrial unfolded protein response (UPR^mt). Such processes fall under the greater rubric of cellular surveillance mechanisms. Here we identify a novel p38 signaling cascade that is required to extend life when the mitochondrial electron transport chain is disrupted in worms, and which is blocked by disruption of the Mitochondrial-associated Degradation (MAD) pathway. This novel cascade is defined by DLK-1 (MAP3K), SEK-3 (MAP2K), PMK-3 (MAPK) and the reporter gene <i>Ptbb-6</i>::<i>GFP</i>. Inhibition of known mitochondrial retrograde responses does not alter induction of <i>Ptbb-6</i>::<i>GFP</i>, instead induction of this reporter often occurs in counterpoint to activation of SKN-1, which we show is under the control of ATFS-1. In those mitochondrial bioenergetic mutants which activate <i>Ptbb-6</i>::<i>GFP</i>, we find that <i>dlk-1</i>, <i>sek-3</i> and <i>pmk-3</i> are all required for their life extension.</p></div

<i>Ptbb-6</i>::<i>GFP</i> marks a new cell surveillance pathway.

<p>(<b>A, B</b>) Among genes known to function epistatically to <i>atfs-1</i> in its role in activating the UPR^mt [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006133#pgen.1006133.ref026" target="_blank">26</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006133#pgen.1006133.ref060" target="_blank">60</a>], only F40F12.7/ <i>cbp-3</i> is also required for <i>Ptbb-6</i>::<i>GFP</i> expression (A). The role of <i>cbp-3</i> in the <i>Ptbb-6</i>::<i>GFP</i> pathway is distinct from its role in the UPR^mt, since ceramide addition only replaces the requirement for <i>cbp-3</i> in UPR^mt activation (B). (<b>C, D</b>) Monoamine neurotransmission and neuromodulation are dispensable for <i>Ptbb-6</i>::<i>GFP</i> activation. Neither dietary supplementation of L-tyramine, octopamine or dopamine (C), nor genetic inactivation of catecholamine synthesis (D), alters <i>Ptbb-6</i>::<i>GFP</i> activation following mitochondrial ETC disruption. (<b>E, F</b>) RNAi-mediated inhibition of core MAD pathway genes strongly inhibit <i>Ptbb-6</i>::<i>GFP</i> induction by <i>isp-1(qm150)</i> worms. Quantitative fluorescence imaging data is provided in panel F. (n = 4–7 worms per condition; asterisks indicate significantly (<i>p<0</i>.<i>025)</i> different relative to vector-treated animals).</p