Measurement of H₂O₂ within living drosophila during aging using a ratiometric mass spectrometry probe targeted to the mitochondrial matrix

Hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) is central to mitochondrial oxidative damage and redox signaling, but its roles are poorly understood due to the difficulty of measuring mitochondrial H<sub>2</sub>O<sub>2</sub> in vivo. Here we report a ratiometric mass spectrometry probe approach to assess mitochondrial matrix H<sub>2</sub>O<sub>2</sub> levels in vivo. The probe, MitoB, comprises a triphenylphosphonium (TPP) cation driving its accumulation within mitochondria, conjugated to an arylboronic acid that reacts with H<sub>2</sub>O<sub>2</sub> to form a phenol, MitoP. Quantifying the MitoP/MitoB ratio by liquid chromatography-tandem mass spectrometry enabled measurement of a weighted average of mitochondrial H<sub>2</sub>O<sub>2</sub> that predominantly reports on thoracic muscle mitochondria within living flies. There was an increase in mitochondrial H<sub>2</sub>O<sub>2</sub> with age in flies, which was not coordinately altered by interventions that modulated life span. Our findings provide approaches to investigate mitochondrial ROS in vivo and suggest that while an increase in overall mitochondrial H<sub>2</sub>O<sub>2</sub> correlates with aging, it may not be causative

Amino-acid imbalance explains extension of lifespan by dietary restriction in Drosophila

Dietary restriction extends healthy lifespan in diverse organisms but reduces fecundity; this is thought to be because of an adaptive reallocation of nutrients from reproduction to somatic maintenance. Here, the nutrients producing the responses of lifespan and fecundity to dietary restriction in Drosophila are identified. Adding essential amino acids to the dietary restriction condition increased fecundity and decreased lifespan; furthermore, addition of methionine alone rescued fecundity

Intermittent exposure of flies to food does not increase their lifespan.

<p>Throughout adult life, Dahomey females were exposed to daily cycles of starvation∶feeding of either 3 h∶21 h or 6 h∶18 h. Neither treatment had any effect on lifespan. During the periods of starvation, flies had access to water only.</p

Model of the relationship between lifespan and DR protocols that reduce access to food either by intermittent exposure (left panel) or nutrient dilution (right panel).

<p>These demonstrate how the composition of food used for intermittent feeding protocols could lead to the false conclusion that DR does not exist for an organism. Three different diets are shown that vary in a given nutrient concentration from ‘very dilute’ to ‘concentrated’. In this example, increasing access to the concentrated diet causes lifespan to rise to a peak (DR) beyond which lifespan decreases. At some point (marked here as the ‘point of satiety’) the animal will no longer be able to eat any more food, meaning the nutrition level it experiences is capped and no further increase in availability will further decrease lifespan. For the dilute and very dilute diets, the point of satiety is reached before the level of nutrients ingested has a chance to cause lifespan to reduce. Thus, there is no lifespan increase for any intermediate level of food restriction, making it look like the organism does not exhibit a DR response. For flies, these problems can be avoided by assaying lifespan in the presence of excess food that is diluted to differing extents. The relationship of this situation to DR by intermittent feeding is represented by taking a cross-section through the graph on the left. The plot on the right shows the type of data presented herein and for other invertebrate studies.</p

The Dahomey genetic background is capable of the longest lifespan and greatest reproductive output of the wild-type strains tested.

<p>For median lifespan, the data are the averages from the longest lived conditions for each strain. For lifetime fecundity they are the average of the condition producing the greatest lifetime reproduction. It should be noted that the conditions under which these occur is different for the two traits, as predicted by the expectations of DR, and that they may be different for each different strain. Data from n independent repeats, where n = 5 for Dahomey, CantonS and OregonR; n = 2 for yw, and; n = 1 for w1118 and wDahomey.</p

Amino-acid imbalance explains extension of lifespan by dietary restriction in Drosophila

Dietary restriction extends healthy lifespan in diverse organisms and reduces fecundity(1,2). It is widely assumed to induce adaptive reallocation of nutrients from reproduction to somatic maintenance, aiding survival of food shortages in nature(3-6). If this were the case, long life under dietary restriction and high fecundity under full feeding would be mutually exclusive, through competition for the same limiting nutrients. Here we report a test of this idea in which we identified the nutrients producing the responses of lifespan and fecundity to dietary restriction in Drosophila. Adding essential amino acids to the dietary restriction condition increased fecundity and decreased lifespan, similar to the effects of full feeding, with other nutrients having little or no effect. However, methionine alone was necessary and sufficient to increase fecundity as much as did full feeding, but without reducing lifespan. Reallocation of nutrients therefore does not explain the responses to dietary restriction. Lifespan was decreased by the addition of amino acids, with an interaction between methionine and other essential amino acids having a key role. Hence, an imbalance in dietary amino acids away from the ratio optimal for reproduction shortens lifespan during full feeding and limits fecundity during dietary restriction. Reduced activity of the insulin/insulin-like growth factor signalling pathway extends lifespan in diverse organisms(7), and we find that it also protects against the shortening of lifespan with full feeding. In other organisms, including mammals, it may be possible to obtain the benefits to lifespan of dietary restriction without incurring a reduction in fecundity, through a suitable balance of nutrients in the diet

Chemical changes in aging Drosophila melanogaster

The “Green Theory” of aging proposes that organismal lifespan is limited by the failure to repair molecular damage generated by a broad range of metabolic processes. Two specific predictions arise from this: (1) that these processes will produce a wide variety of stable but dysfunctional compounds that increase in concentration with age, and (2) that organisms maintained under conditions that extend lifespan will display a reduced rate of accumulation of such “molecular rubbish”. To test these predictions, novel analytical techniques were developed to investigate the accumulation of damaged compounds in Drosophila melanogaster. Simple preparative techniques were developed to produce digests of whole D. melanogaster for use in three-dimensional (3D) fluorimetry and 1H NMR spectrometry. Cohorts of Drosophila maintained under normal conditions showed an age-related increase in signals consistent with damage whereas those maintained under conditions of low temperature and dietary restriction did not. 1H NMR revealed distinct age-associated spectral changes that will facilitate the identification of novel compounds that both increase and decrease during aging in this species. These findings are consistent with the predictions of the “Green Theory”