Norms of reaction of wild-derived <i>Drosophila</i> populations.

<p>Lifespan and fecundity showed different shapes of response to yeast concentration. The effect of yeast concentration on lifespan best fitted a third-order polynomial equation (non-linear regression) and on fecundity a linear equation (linear regression). Goodness of fit is high in both models, as represented by R² values: For mean lifespans: 0.88, 0.74, 0.80, 0.88, 0.79 for <i>W^Dah</i>, <i>FRA</i>, <i>GRE</i>, <i>GER</i>, <i>NETH</i> strains respectively. For fecundity: 0.90, 0.87, 0.87, 0.89, 0.91 for <i>W^Dah</i>, <i>FRA</i>, <i>GRE</i>, <i>GER</i>, <i>NETH</i> strains respectively. For mean lifespans, best-fit values ranged for B0: 6.6 to 28, for B1: 86.5 to 176, for B2: -81.6 to -151, for B3: 20.9 to 36.8. Linear regression analysis for fecundity: for <i>W^Dah</i>: F = 484.2, DFn = 1, DFd = 48, <i>p</i><1×10⁻⁴, for <i>FRA</i>: F = 335.7, DFn = 1, DFd = 48, <i>p</i><1×10⁻⁴, for <i>GRE</i>: F = 309.9, DFn = 1, DFd = 48, <i>p</i><1×10⁻⁴, for <i>GER</i>: F = 373.6, DFn = 1, DFd = 48, <i>p</i><1×10⁻⁴, for <i>NETH</i>: F = 508.2, DFn = 1, DFd = 48, <i>p</i><1×10⁻⁴. Lifespan curves and fecundity slopes differed significantly among strains (for lifespan curves: F = 63.23, DFn = 16, DFd = 230, <i>p</i><1×10⁻⁴, for fecundity slopes: F = 27.10, DFn = 4, DFd = 240, <i>p</i><1×10⁻⁴).</p

Effect of DR on median and maximum lifespan of <i>Drosophila melanogaster</i> wild-derived strains.

<p>A yeast concentration range of 0.1 to 2.0 captured the DR response of all strains. Both median and maximum lifespans were affected by yeast concentration, in all cases. Maximum lifespan for <i>W^Dah</i>: F = 114.5, R² = 0.9106, <i>p</i><1×10⁻⁴, for <i>FRA</i>: F = 52.76, R² = 0.8242, <i>p</i><1×10⁻⁴, for <i>GRE</i>: F = 58.22, R² = 0.8381, <i>p</i><1×10⁻⁴, for <i>GER</i>: F = 91.41, R² = 0.8904, <i>p</i><1×10⁻⁴, for <i>NETH</i>: F = 48.92, R² = 0.8130, <i>p</i><1×10⁻⁴, one-way ANOVA test, <i>n</i> = 100). Maximum lifespan was calculated as the average lifespan of the most long-lived 10% of flies.</p

Lifespan and fecundity of wild–derived <i>Drosophila melanogaster</i> strains responded to DR.

<p>The mean lifespans of all wild-derived strains and the laboratory strain exhibited a tent-shaped response to DR with highest mean lifespan values at 0.5 or 1.0 yeast concentration. Female fecundity showed a monotonic increase with yeast concentration in all strains. Both mean lifespan and fecundity were significantly affected by food. Mean lifespan for <i>W^Dah</i>: F = 94.21, R² = 0.8933, <i>p</i><1×10⁻⁴, for <i>FRA</i>: F = 35.42, R² = 0.7589, <i>p</i><1×10⁻⁴, for <i>GRE</i>: F = 81.23, R² = 0.8784, <i>p</i><1×10⁻⁴, for <i>GER</i>: F = 96.66, R² = 0.8957, <i>p</i><1×10⁻⁴, for <i>NETH</i>: F = 43.25, R² = 0.7936, <i>p</i><1×10⁻⁴, one-way ANOVA test. Fecundity for <i>W^Dah</i>: F = 136.5, R² = 0.9239, <i>p</i><1×10⁻⁴, for <i>FRA</i>: F = 158.3, R² = 0.9337, <i>p</i><1×10⁻⁴, for <i>GRE</i>: F = 103.1, R² = 0.9016, <i>p</i><1×10⁻⁴, for <i>GER</i>: F = 107.6, R² = 0.9054, <i>p</i><1×10⁻⁴, for <i>NETH</i>: F = 123.5, R² = 0.9165, <i>p</i><1×10⁻⁴, one-way ANOVA test. Points: mean lifespan. Bars: estimate of mean number of eggs laid/fly/day ± standard error; connected points: mean lifespan in days (<i>n</i>  = 100). Data shown are from a single trial with second generation flies of the wild-derived strains.</p

Wild-derived strain <i>NETH</i> had similar greatest mean lifespan/fecundity values to laboratory strain <i>W^Dah.</i>

<p>Under all food conditions tested in this study, the combined greatest mean lifespan and greatest mean fecundity values were seen in the laboratory-adapted <i>W^Dah</i> and wild-derived <i>NETH</i> strains, compared to each of the other strains (for lifespan: <i>p</i><0.001, log rank test, for fecundity: <i>p</i><0.01, one-way ANOVA with Bonferroni's Multiple Comparison test). Greatest mean lifespans for <i>GRE</i>, <i>GER</i> and <i>FRA</i> strains were obtained from 0.5 yeast concentration food, while for <i>W^Dah</i> and <i>NETH</i> strains from 1.0 yeast concentration. For all strains greatest fecundity occurred at 2.0 yeast concentration food. Wild-derived strain <i>NETH</i> had similar greatest mean lifespan/mean fecundity values to the laboratory strain <i>W^Dah</i> (for lifespan: <i>p</i>>0.05, log rank test, for fecundity: <i>p</i>>0.05, one-way ANOVA with Bonferroni's Multiple Comparison test). Data are derived from results shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074681#pone-0074681-g001" target="_blank">Figures 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074681#pone-0074681-g002" target="_blank">2</a>. Error bars indicate standard deviation (SD).</p

<i>Drosophila melanogaster</i> wild-derived strains used in this study.

<p><i>Drosophila melanogaster</i> wild-derived strains used in this study.</p

Effects of metformin on female egg-laying and post-reproductive survival.

<p>(<b>A</b>) Egg-laying profiles of wild-type females treated with 0 mM, 5 mM, 10 mM, 25 mM, 50 mM and 100 mM metformin. Eggs were counted from 10 vials per treatment (10 females per vial) over a 24 hour period after 7, 14 and 21 days of metformin treatment. Data are shown as means ±SEM. * denotes statistically significant difference (<i>P</i><0.05). No significant differences were observed in egg-laying between females on 0 mM, 5 mM and 10 mM metformin at any time point. After 7 days of treatment, females on 25 mM and 50 mM metformin laid significantly more eggs than untreated controls. After 14 days of treatment, females on 50 mM laid significantly fewer eggs than untreated controls. Females on 100 mM metformin laid significantly fewer eggs than untreated controls at all time points. (<b>B</b>) Survival curves for post-reproductive wild-type females maintained on food containing no metformin or final concentrations 25 mM and 50 mM metformin. Flies (n = 250 for each concentration) were switched onto food containing metformin at 39 days of age (red arrow). Females maintained on 25 mM or 50 mM showed reduced survival compared to flies maintained in the absence of metformin (<i>P</i><0.001 by the Log-rank test).</p

Metformin treatment of adult <i>Drosophila</i> activates AMPK. A.

<p>Mass spectrometric determination of metformin concentration in whole fly extracts. Female flies were sampled after 7 days of metformin treatment at concentrations of 1, 10 and 100 mM. Before sampling, flies were incubated in the absence of metformin for 5 hours to allow for gut emptying. A dose-dependent increase in metformin accumulation in fly tissues was observed. Data are represented as the mean of three independent replicate samples ± SEM. <b>B.</b> Western blot analysis of phospho-Thr172-AMPK expression in whole-fly protein extracts. Flies were sampled after 7 days of metformin treatment at concentrations of 0, 5, 10, 25, 50 and 100 mM. A dose-dependent increase in phospho-Thr172-AMPK levels was observed. Actin was used as a loading control.</p

Metformin does not increase lifespan in <i>Drosophila.</i> A.

<p>Survival curves of wild-type (<i>Dahomey</i>) males and females maintained on food containing no metformin or final concentrations of 1 mM, 2.5 mM or 5 mM metformin. No significant differences in survival were observed between metformin treated flies and non-treated controls by the Log-rank test. For males, median survival times on 0 mM, 1 mM, 2.5 mM and 5 mM metformin were 57 (n = 96), 59 (n = 96), 55 (n = 97) and 57 (n = 96) days, respectively. For females, median survival times were 67 days for all conditions (0 mM n = 96, 1 mM n = 96, 2.5 mM n = 99 and 5 mM n = 91). <b>B.</b> Survival curves of wild-type (<i>Dahomey</i>) males and females maintained on food containing no metformin or final concentrations of 5 mM, 10 mM, 25 mM, 50 mM or 100 mM metformin. The survival curves for males maintained on 0 mM, 5 mM, 10 mM, 25 mM and 50 mM are not significantly different, while males maintained on 100 mM metformin were significantly shorter lived than non-treated controls (<i>P</i><0.0001 by the Log-rank test). Median survival times for males on 0 mM, 5 mM, 10 mM, 25 mM, 50 mM and 100 mM metformin were 54 (n = 97), 58 (n = 91), 51 (n = 93), 51 (n = 93), 51 (n = 98) and 37 (n = 99) days, respectively. The survival curves for females maintained on 0 mM, 5 mM and 10 mM metformin are not significantly different. Females maintained on 25 mM, 50 mM and 100 mM metformin were significantly shorter lived than non-treated controls (<i>P</i><0.001 by the Log-rank test). Median survival times for females on 0 mM, 5 mM, 10 mM, 25 mM, 50 mM and 100 mM metformin were 65 (n = 96), 65 (n = 100), 63 (n = 98), 58 (n = 101), 51 (n = 91) and 22 (n = 99) days respectively.</p

Metformin reduces lipid stores and causes intestinal fluid imbalance.

<p>(<b>A</b>) Quantitation of triacylglycerides (TAGs) in flies treated with 0 mM, 1 mM, 10 mM and 100 mM metformin for 7 days. TAG levels decrease with increasing metformin concentration with significantly lower levels in the 10 mM and 100 mM metformin groups compared to untreated controls (<i>P</i><0.05, n = 10 (2 flies per replicate)). (<b>B</b>) Metformin treatment of female flies does not affect the number of fecal deposits produced per fly over a 24 hour period (<i>P>0.05</i>, Wilcoxen test, n = 5 (5 flies per replicate)). (<b>C</b>) Metformin treatment of female flies does not affect the size of fecal deposits as measured by the mean area of deposits (<i>P>0.05</i>, Wilcoxen test, n = 5 (5 flies per replicate)). (<b>D</b>) Female flies fed with 100 mM metformin produce more concentrated fecal deposits as measured by increased average dye intensity (<i>P</i><0.05, Wilcoxen test, n = 5 (5 flies per replicate)). (<b>E</b>) Female flies fed with 100 mM metformin produce more RODs as a percentage of their total excreta output (<i>P</i><0.05, Wilcoxen test, n = 5 (5 flies per replicate)).</p

Comparative survival characteristics of female wild type (WT) and <i>Irs1^−/−</i> mice.

<p>Lifespan is reported in days (± s.e.m., where appropriate) for WT and <i>Irs1^−/−</i> mice from this current study and our original study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016144#pone.0016144-Selman2" target="_blank">[12]</a>. Combined = Combined lifespan data derived from current study and original study. N = sample size.</p