Results: 4

1.
Figure 1

Figure 1. Metformin treatment of adult Drosophila activates AMPK. A.. From: Activation of AMPK by the Putative Dietary Restriction Mimetic Metformin Is Insufficient to Extend Lifespan in Drosophila.

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. 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.

Cathy Slack, et al. PLoS One. 2012;7(10):e47699.
2.
Figure 4

Figure 4. Metformin reduces lipid stores and causes intestinal fluid imbalance.. From: Activation of AMPK by the Putative Dietary Restriction Mimetic Metformin Is Insufficient to Extend Lifespan in Drosophila.

(A) 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 (P<0.05, n = 10 (2 flies per replicate)). (B) Metformin treatment of female flies does not affect the number of fecal deposits produced per fly over a 24 hour period (P>0.05, Wilcoxen test, n = 5 (5 flies per replicate)). (C) Metformin treatment of female flies does not affect the size of fecal deposits as measured by the mean area of deposits (P>0.05, Wilcoxen test, n = 5 (5 flies per replicate)). (D) Female flies fed with 100 mM metformin produce more concentrated fecal deposits as measured by increased average dye intensity (P<0.05, Wilcoxen test, n = 5 (5 flies per replicate)). (E) Female flies fed with 100 mM metformin produce more RODs as a percentage of their total excreta output (P<0.05, Wilcoxen test, n = 5 (5 flies per replicate)).

Cathy Slack, et al. PLoS One. 2012;7(10):e47699.
3.
Figure 2

Figure 2. Metformin does not increase lifespan in Drosophila. A.. From: Activation of AMPK by the Putative Dietary Restriction Mimetic Metformin Is Insufficient to Extend Lifespan in Drosophila.

Survival curves of wild-type (Dahomey) 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. Survival curves of wild-type (Dahomey) 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 (P<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 (P<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.

Cathy Slack, et al. PLoS One. 2012;7(10):e47699.
4.
Figure 3

Figure 3. Effects of metformin on female egg-laying and post-reproductive survival.. From: Activation of AMPK by the Putative Dietary Restriction Mimetic Metformin Is Insufficient to Extend Lifespan in Drosophila.

(A) 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 (P<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) 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 (P<0.001 by the Log-rank test).

Cathy Slack, et al. PLoS One. 2012;7(10):e47699.

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