compared to control flies
As shown before puml1 flies have an increased starvation resistance and store more TAGs than control flies. However, a direct hydrolytic activity on lipids (TAGs and DAGs) could not be detected. Perhaps, this is caused by a lower mobilization rate of storage lipid. In order to test of if lipid mobilization was impaired in puml1 flies a starvation assay was performed. Compared to bmm1, the general capability to mobilize storage lipids was not impaired in puml1during starvation (Figure 21).
Figure 21 puml1 flies mobilize lipids faster during starvation than control flies. Plotted are means of TAG± SEM. Both, puml1 and bmm1 exhibit increased body fat storage at starvation start (six days after eclosure; one-way ANOVA F(2,8)=19.03, P=9.09^10-4). During starvation, all tested flies could mobilize
storage lipids (two-way ANOVA, genotype and starvation time as fixed effects, F(2,31)=72.41, P=4.23^10-14). bmm1 showed a slower TAG mobilization during the first 24h of starvation and a substantial amount of TAGs remained immobilized in dead flies. puml1 mobilized around half of the storage lipids (much more then control flies) within the first 24h of starvation. Lipid utilization became less in the following time of starvation and a significant amount of lipids remained in dead flies compared to control flies (one-way ANOVA, F(2,14)=5.40, P=0.01; Fisher`s LSD P<0.05). Control flies exhibited a similar pattern of reduced lipid mobilization with prolonging starvation but nearly used up all stored lipids.
Indeed, puml1 flies used up ~30% more lipids during the first 24h of starvation compared to control flies. Besides a significantly higher amount of un-mobilized lipids found in post-mortem flies, in total puml1 utilized on average ~60% more lipids than control flies during starvation time. Thus, puml1 is not crucial for lipid utilization in general. Nevertheless, it is possible that a subset of lipids is not mobilized in puml1 flies.
Figure 22 Locomotor activity and metabolic rate of puml1 are similar to control flies during starvation.
(A) For each fly line, the individual locomotor activity levels of individual flies (n=64) were measured in 5-minute bins. In order to generate a group profile, the sum of activity was plotted for each genotype. (B) Box plot for the total number of beam-passes during starvation time per animal;
Center lines show the median, box limits indicate 25th and 75th percentiles as determined by OriginPro software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; n= 63 for each genotype. Average of total beam-passes during starvation was significantly higher in bmm1 flies (Mann-Whitney test; ***=P<0.001). (C) Metabolic rate is represented as box plot (generated as described shortly before). Metabolic rate was
lower in starved (16 hours under starvation) cohorts (one cohort= 3 animals; checked at zeitgeber +8h) but no difference between puml1 and control flies could be detected (Mann-Whitney test).
3.5.2 Total locomotor activity is not increased in pummelig mutant flies under starvation.
In order to identify the cause for the increased lipid consumption in puml1 flies, locomotor activity form starving flies was monitored in the Drosophila activity monitoring system (Trikinetics). Single flies were kept in glass tubes and motion was quantified by counting passes through an infrared light beam. There was no difference in the activity between puml1 and control flies during starvation (Figure 22). Activity peaks were similar between all tested genotypes. Total starvation activity (Figure 22) was significantly higher in bmm1 compared to puml1 and control flies. Since median survival time under starvation (Figure 18) is increased in bmm1 flies, allowing for a higher total activity, average total accumulated activity during starvation was increased in bmm1 flies. However, higher lipid utilization of puml1 flies, especially in the first twenty-four hours, was not correlated with higher activity during starvation.
3.5.3 Metabolic rate is not changed in pummelig mutant flies
Modulation of lipid storage in puml1 is different compared to control flies. During ad libitum feeding puml1 have increased body fat storage. Under fasting conditions these lipids are utilized faster than in control flies but locomotor activity is not changed. A possible reason for the higher lipid consumption might be a different regulation of metabolic rate in puml1. In order to address this, a metabolic rate assay was performed using fed and fasted (16h under starvation) flies. Metabolic rate was significantly reduced under starvation compared to ad libitum fed flies (Figure 22).
However, no difference in metabolic rate between puml1 and control flies was detected for fed as well as for fasted flies (Figure 22).
3.5.4 Glycogen storage is decreased in pummelig mutant flies
As shown before puml1 had higher lipid storage under fed conditions but faster utilization of this energy storage. puml1 flies did not have a higher activity under starvation and metabolic rates of were not different under feeding condition or during nutrient deprivation. As only the oxygen consumption was monitored for the
metabolic activity and no data for CO2 was acquired, the oxidative quotient could not be used to identify which energy source is mainly used by the fly. Therefore, a more detailed time course experiment was performed to monitor energy stores during the first 24h of starvation. Besides neutral lipids also protein content and glycogen stores were analysed (Figure 23). The amount of protein did not change significantly during the first 24h of starvation, whereas lipids were utilized faster in puml1 flies.
Interestingly, glycogen stores in puml1 were decreased by ~42% compared to control flies (Student`s test, P=7,51*10-4) in fed flies. Glycogen stores depleted simultaneously with the lipids in puml1 and control flies (Figure 23). Due to the lower amount of glycogen in puml1 they were used up much faster leaving up lipids as the main energy source. Therefore, higher lipid mobilization of puml1 is mainly caused by lower availability of carbohydrates under nutrient deprivation.
Figure 23 Less glycogen is stored in puml1 flies. Plotted are mean values for Protein (triangle; green), Triacylglycerides (dots; blue) and Glycogen (rectangle, red) ±SEM during starvation. puml1 flies (B) store increased amounts of TAG and have lower Glycogen stores (control flies (A) store ~42% more Glycogen). Protein amounts were not changed significantly during the initial 24h of starvation.
Glycogen stores in puml1 flies (B) were completely utilized during the observation time leaving lipids as the remaining major energy source.
3.5.5 Desiccation resistance is impaired in pummelig mutant flies
It is known that glycogen vastly contributes to desiccation resistance in Drosophila melanogaster (Marron et al., 2003). Therefore, it was tested if the reduced glycogen storage has a negative impact on the mean survival time under these conditions. In correlation with lower Glycogen storage in puml1 also mean survival time under desiccation was significantly decreased (-17% see Figure 24).
Figure 24 Desiccation resistance is decreased in puml1 flies(n=128 animals per genotype; Log Rank test; P=5.41*10-6).
Taken together, it was assumed that the TAG over-storage phenotype in puml1 flies was caused by an impaired mobilization of lipids. Interestingly, conversely to the high TAG storage, Glycogen stores in puml1 flies were significantly lower in puml1 indicating that the storage type of energy in puml1 was changed. In general storage lipid mobilization was not impaired in puml1 flies and metabolic rate was indifferent from control flies (under fed conditions and during starvation). However, puml1 utilized always more lipids during starvation than control flies indicating that lipids might be the preferred substrate for energy generation. The substrate screen identified Puml as an active phospholipase but how this effects lipid storage needs to be unravelled.