P,0.60.036 p,0.60.073 p = 0.Redox Ratio Decreases when Flies are Exposed to Short StarvationThe redox ratio (NAD+/NADH) decreases during starvation, which was first shown by Williamson et al. [27]. During starvation, metabolism switches to storage energy utilization, including b?oxidation of fatty acid, which leads to the generation of ketone bodies by excess level of Acetyl-CoA. Theses changes result in a reduction of the redox ratio, which was first shown by measuring, rather indirectly, the ratio of reactant and product of dehydrogenase reaction [27]. A number of dehydrogenase reactions were known to be at or near equilibrium such as Malate dehydrogenase (EC 1.1.1.37, Mdh). The ratio between free 
concentrations, [NAD+]/[NADH], can be calculated when [malate]/[oxaloacetate] and the equilibrium constant are known [27]. Here using the enzyme recycling assay directly measuring total amount of pyridine nucleotides, we show that the ratio NAD+/NADH decreased under starvation. Fifteen newly eclosed male flies of the w1118 strains were collected and aged to day 10 on 10 yeast, 10 sugar and 2 (w/v) agar food vials. Vials were kept in 25uC, 
60 relative humidity incubator with a 12:12 hours light cycle (light-on at 8:00 a.m.). At 12:00 a.m. on the 10th day, they were transferred to vials containing 2 (w/v) agar, which cannot be utilized as a foodSix samples of 15 male D. melanogaster adults were homogenized in 250 ul homogenization buffer. Following a 5 min 160006g centrifugation, the supernatant was then divided into 3 parts. One part was kept as control. The other two parts were treated with equal volume of chloroform and phenolchloroform respectively. Three parts were then assayed for NAD+ and NADH in duplicates. The concentration of NAD+ and NADH, with S.E.M, are standardized by the concentration of soluble protein inhibitor measured from the control group. The difference in NAD+ and NADH concentration is tested using two sample t-test. See supplementary material for the method of testing redox ratio difference between groups. doi:10.1371/journal.pone.0047584.tsource by Drosophila, and assayed for NAD+, NADH, NADP+ and NADPH after 10 hours. Starting starvation treatment at Zeitgeber time +16 hours is to Autophagy minimize food intake variation among animals, as it was clearly demonstrated by Xu et al. that feeding activity is at its minimal at this time [32].The control group was kept on the aforementioned yeast-sugar-agar food for the same amount of time. As summarized in table 3, we found that NAD+/NADH redox ratio of well-fed Drosophila is around 8, and with 10 hours of starvation the redox ratio decreases to about 4 which is highly significant. The ratio of total NADP+/NADPH was found to be around 0.2. It also decreases after 10 hours of starvation (Table 3). We verified the animals were truly in a starvation state by measuring the level of triacylglyceride, glycogen and glucose and detected their levels in the starved group were significantly lower (Fig. 5). We found the concentration of protein is not significantlyFigure 4. Comparison of three different extraction methods. Three sets of 15 D. melanogaster adult males were subjected to different treatments. Homogenized in buffer with additional 6 M guanidine-HCl or: homogenized and treated with equal volume phenol-chloroform or chloroform only. The samples were then assayed for NADx in duplicates. doi:10.1371/journal.pone.0047584.gMeasuring Redox Ratio by a Coupled Cycling AssayTable 3. The level of.P,0.60.036 p,0.60.073 p = 0.Redox Ratio Decreases when Flies are Exposed to Short StarvationThe redox ratio (NAD+/NADH) decreases during starvation, which was first shown by Williamson et al. [27]. During starvation, metabolism switches to storage energy utilization, including b?oxidation of fatty acid, which leads to the generation of ketone bodies by excess level of Acetyl-CoA. Theses changes result in a reduction of the redox ratio, which was first shown by measuring, rather indirectly, the ratio of reactant and product of dehydrogenase reaction [27]. A number of dehydrogenase reactions were known to be at or near equilibrium such as Malate dehydrogenase (EC 1.1.1.37, Mdh). The ratio between free concentrations, [NAD+]/[NADH], can be calculated when [malate]/[oxaloacetate] and the equilibrium constant are known [27]. Here using the enzyme recycling assay directly measuring total amount of pyridine nucleotides, we show that the ratio NAD+/NADH decreased under starvation. Fifteen newly eclosed male flies of the w1118 strains were collected and aged to day 10 on 10 yeast, 10 sugar and 2 (w/v) agar food vials. Vials were kept in 25uC, 60 relative humidity incubator with a 12:12 hours light cycle (light-on at 8:00 a.m.). At 12:00 a.m. on the 10th day, they were transferred to vials containing 2 (w/v) agar, which cannot be utilized as a foodSix samples of 15 male D. melanogaster adults were homogenized in 250 ul homogenization buffer. Following a 5 min 160006g centrifugation, the supernatant was then divided into 3 parts. One part was kept as control. The other two parts were treated with equal volume of chloroform and phenolchloroform respectively. Three parts were then assayed for NAD+ and NADH in duplicates. The concentration of NAD+ and NADH, with S.E.M, are standardized by the concentration of soluble protein measured from the control group. The difference in NAD+ and NADH concentration is tested using two sample t-test. See supplementary material for the method of testing redox ratio difference between groups. doi:10.1371/journal.pone.0047584.tsource by Drosophila, and assayed for NAD+, NADH, NADP+ and NADPH after 10 hours. Starting starvation treatment at Zeitgeber time +16 hours is to minimize food intake variation among animals, as it was clearly demonstrated by Xu et al. that feeding activity is at its minimal at this time [32].The control group was kept on the aforementioned yeast-sugar-agar food for the same amount of time. As summarized in table 3, we found that NAD+/NADH redox ratio of well-fed Drosophila is around 8, and with 10 hours of starvation the redox ratio decreases to about 4 which is highly significant. The ratio of total NADP+/NADPH was found to be around 0.2. It also decreases after 10 hours of starvation (Table 3). We verified the animals were truly in a starvation state by measuring the level of triacylglyceride, glycogen and glucose and detected their levels in the starved group were significantly lower (Fig. 5). We found the concentration of protein is not significantlyFigure 4. Comparison of three different extraction methods. Three sets of 15 D. melanogaster adult males were subjected to different treatments. Homogenized in buffer with additional 6 M guanidine-HCl or: homogenized and treated with equal volume phenol-chloroform or chloroform only. The samples were then assayed for NADx in duplicates. doi:10.1371/journal.pone.0047584.gMeasuring Redox Ratio by a Coupled Cycling AssayTable 3. The level of.