Overexpression of Sir2 in the adult fat body is sufficient to extend lifespan of male and female Drosophila

Moderate Overexpression of Sir2 in the Fat Body Elongates Lifespan of Drosophila

To avoid differences in genotypes between control and experimental animals, we used the inducible GeneSwitch System [20] to overexpress Sir2 in the fat body of adult flies. Here, tissue-specific overexpression of Sir2 is achieved by administration of mifepristone (RU486) to the F1 generation of a cross of UAS-Sir2 with the driver line S₁106-Gal4 while genetically and developmentally identical controls feed on normal medium supplemented with the same dose of the diluent ethanol. We first examined the expression level of Sir2 under the experimental conditions. UAS-Sir2>S₁106-Gal4 flies were fed 400 μM RU486 or diluent only for 24 h. Fat bodies were dissected and qPCR for Sir2 mRNA was performed. We detected an approx. 3.5 fold and 2.5 fold up-regulation of Sir2 gene expression in the fat bodies of females and males, respectively (Fig. S1). This system induces a moderate increase of Sir2 expression, which is presumably in the physiological range of expression changes.

Until recently, studies that aimed to investigate the effect of Sir2 on lifespan mainly manipulated its gene expression in whole animals or the nervous system. We decided to study the effect of overexpressing Sir2 in the fat body as it is strongly expressed in this tissue [3]. Moreover, the fat body integrates the response to both immune and metabolic stimuli, which are both important modulators of the aging process [21, 22]. To do so, we determined lifespan of mated UAS-Sir2>S₁106-Gal4 flies fed on medium with or without RU486 throughout adult life. Survival curves of females (Fig. 1A) and males (Fig. 1C) overexpressing Sir2 are significantly shifted to the right. As shown in Table 1, median lifespan of females and males treated with RU486 is increased by 12 % and 13 % compared to controls, respectively. These data show that even the relatively weak overexpression of Sir2 induced by the S₁106-Gal4 line in the adult fat body promotes longevity in flies, largely by delaying the onset of cohort aging. To rule out the possibility that the GAL4 activator RU486 itself may elongate lifespan of Drosophila, we performed lifespan experiments with flies from a crossing of the w¹¹¹⁸ strain and the driver line S₁106-Gal4. Flies were treated as described above and no significant shift in survival curves was observed for either females (Fig. 1B) or males (Fig. 1D) compared to controls.

Figure 1. Increased expression of Sir2 in the fat body extended lifespan of male and female flies. Mated female (A) or male (C) UAS-Sir2>S₁106-Gal4 flies were fed 400 μM RU486 or diluent (ethanol) throughout adult life and lifespan was determined. As a control for a potential lifespan extending effect of RU486, the driver line was crossed to w¹¹¹⁸ and female (B) or male control flies (D) of the F1 generation were treated as the experimental flies. For p-values of log rank test see Table 1, n = 100 for each group.

Gene Expression Profiles Induced by Sir2 Overexpression and Dietary Restriction Do not Match

The finding that fat body Sir2 can promote a long life led us to examine which candidate mechanisms may play a role in conferring Sir2-dependent longevity. As several studies suggest that Sir2 mediates at least parts of the response to dietary restriction (DR)[3, 17] we aimed to elucidate how these two interventions alter gene expression patterns of the fat body and to what extent transcriptional responses match. We performed microarray analysis with RNA isolated from fat bodies of mated, age-matched females either subjected to DR for 7 d or with fat body specific Sir2 overexpression after 24 h induction by RU486. The control and DR food was prepared according to recommendations by Ja et al. [23] to prevent desiccation, which robustly resulted in prolonged lifespan of male and female w¹¹¹⁸ flies (Fig. S2). Surprisingly, when comparing gene expression profiles we noticed that only roughly 10 % of up- (p value 0.355) or down-regulated genes (p value 0.383) overlap between both samples (Fig 2A, B). This is a surprising small overlap, indicative for almost completely independent processes, especially when taking into account that fat tissue of age- and gender-matched flies was used for both analyses.

Figure 2. Fat body Sir2 does not play a major role in the DR response. Transcriptional profiles of fat bodies from either w1118 females after a 7 d DR period or UAS-Sir2>S₁106-Gal4 females after 24 h of Sir2 overexpression were compared. The scaled Venn diagrams depict the actual relationship of the numbers of up-regulated (A) and down-regulated genes (B) of both samples and the number of overlapping genes. Only roughly 10 % of differentially expressed genes are overlapping between DR samples and Sir2 overexpression. Statistical analyses were performed with Fisher's exact test. Genes, which were up-or downregulated > 1.5 fold in at least 2 of 3 microarrays were included in the diagrams shown. Hierarchical clustering of DR and Sir2 Arrays reveals that transcriptional patterns elicited by DR and Sir2 overexpression in the fat body do not show substantial similarities. (D) mRNA levels of Sir2 in female w¹¹¹⁸ whole animals and in their fat bodies were unchanged after DR. Shown are mean values of 3 replicates + SEM.

Furthermore, hierarchical clustering of the microarray data shows substantial differences in gene expression patterns induced by both interventions (Fig 2C). Especially genes down-regulated in DR sets are up-regulated in Sir2 gene sets. Additionally, we did not detect an increase of Sir2 transcription in response to DR as reported by other groups, neither in whole animals nor in their fat tissues (Fig. 2D). Taken together, these results imply that fat body Sir2 evokes other cellular processes than those elicited by DR.

Fat Body Sir2 Regulates Lipid Droplet Proteome Associated Genes in a Gender-specific Manner

Based on the transcriptome studies performed with isolated fat tissue from Sir2 overexpressing males and females, we focused on those genes that are relevant for fat tissues. Within this tissue lipid droplets are of central importance to regulate fat utilization [24]. Thus, we hypothesized that fat body Sir2 modulates the composition of the lipid droplet proteome and compared the gene expression data from fat bodies overexpressing Sir2 with a list of Gene IDs corresponding to the lipid droplet proteome as defined by Beller et al. [25]. Corresponding genes of 39 out of 254 lipid droplet associated proteins were found to be differentially regulated in fat bodies overexpressing Sir2 which corresponds to 15 % of the lipid droplet proteome (Fig. 3A; p value 0.007). The (putative) functions of these genes are very diverse and range from lipid storage and fatty acid metabolism, stress response and energy metabolism, to chromatin assembly (Table 2).

Figure 3. Gene expression profiles of fat bodies overexpressing Sir2 show notable intersections with the lipid droplet proteome. (A) Differentially regulated genes (up-and down-regulated) found in Sir2 overexpressing males and females were compared with the lipid droplet proteome as defined by Beller et al. [25]. The Venn diagram shows considerable intersections between these different sample types (statistical analyses were performed with Fisher's exact test; see Table 2 for a list of intersecting genes). (B) To view gender-specific effects, the transcriptional profiles of fat bodies from UAS-Sir2>S₁106-Gal4 males and females after 24 h of Sir2 overexpression were compared. The scaled Venn diagrams depict the actual relationship of the numbers of up- and down-regulated genes of both samples and the number of overlapping genes. Genes which were up- or down-regulated > 1.5 fold in at least 2 of 3 microarrays were included in the diagrams shown in (A) and (B).

When comparing gene expression patterns of male and female fat bodies overexpressing Sir2 we found great differences between genders. Only 40 of 619 genes up-regulated in females were also found within the 320 genes up-regulated in males (Fig. 3B, p value 0.123). This overlap corresponds to 6.5 % and 12.5 % of up-regulated genes in females and males, respectively. The situation was different for down-regulated genes where 11 % and 22 % were overlapping between females and males, respectively (p value < 0.0001). These gender-specific differences are mirrored in the comparison of genes affected by Sir2 overexpression and the genes corresponding to proteins associated with lipid droplets. Considering the data for males and females separately 17 (7 %) and 25 (10 %) of the lipid droplet proteome genes are regulated, respectively (Fig. 3A). But only 1 gene (Glutamate oxaloacetate transaminase 2) is up-regulated in both (Table 2) whereas all other genes are not or inversely regulated in the respective other gender. Taken together, these results point to a role for fat body Sir2 in modulating the composition of the lipid droplet proteome, which is obviously gender-specific.

Fat Body Sir2 Increases Starvation Resistance in Male Flies

The fat body is the major storage organ of the fly, which regulates the response to differing nutrient conditions. Sir2 is capable of sensing cellular energy levels, as its enzymatic activity is NAD⁺- dependent [26]. We hypothesized that fat body Sir2 participates in regulating the response to changes in nutrient conditions when promoting longevity. One measure for the ability to react to changing nutrient supply is to determine starvation resistance. Mated UAS-Sir2>S₁106-Gal4 flies fed on 1 % agar with or without RU486 and starvation medium was changed every 2-3 days. Dead flies were counted 1-3 times a day. Fat body Sir2 appeared to have no impact on female starvation resistance as median lifespan of control and Sir2 overexpressing flies showed no difference (Fig. 4A, C). However, fat body Sir2 increased starvation survival in males by 13 % (Fig. 4B, C). In line with the transcript-tome analysis we found a small gender-specific reaction to starvation after Sir2 overexpression in the fat body.

Figure 4. Fat body Sir2 increases starvation survival in males. Mated female (A) or male (B) UAS-Sir2>S₁106-Gal4 flies fed on 1 % agar containing 400 μM RU486 or diluent (ethanol) and dead flies were scored 1-3 times a day. (C) Median lifespan of females was 217 ± 6 h and 215 ± 1 h in control and experimental flies, respectively. For males median lifespans were 172 ± 4 h and 185 ± 7 h for control and Sir2 overexpressing flies, respectively. Data are represented as means + SEM. Log rank analysis was performed using GraphPad Prism 6.00, n = 50.

No Influence of Sir2 on Fat Storage and Fat Cell Morphology

As Sir2 overexpression in the fat body did not only extend lifespan of the flies but also influenced the relative abundance of lipid droplet proteome associated genes and starvation resistance in a gender-specific manner we wanted to elucidate the role of fat body Sir2 on fat storage and morphology of the fat cells. We tested if Sir2 overexpression affects relative body fat content, body weight, the morphology of the fat cells and the size of the fat droplets in males and females.

Mated UAS-Sir2>S₁106-Gal4 males or females were fed on medium containing RU486 or ethanol for 7 d before they were weighed and relative body fat content was quantified by a coupled colorimetric assay according to Hildebrandt et al. [27]. Neither sex showed fat body Sir2 - dependent effects on fat storage (Fig. 5A) and body weight (Fig. 5B). To analyze changes in the morphology of the fat body we performed Bodipy staining with L3 larvae (Fig. 5 D, G), adult females (Fig. 5 E, H) and males (Fig. 5 F, I). Although fat cell size and filling levels of fat stores obviously varied substantially between all sample types, no effect of Sir2 overexpression (Fig. 5 D-I, bottom row) was observed on these parameters compared to controls (Fig. 4 D-I, top row). We randomly selected 14 pictures comprising 7 biological replicates and measured diameters of fat droplets of the larval samples (Fig. 5C) to confirm the results of the qualitative analysis of the tissues. Altogether, these results do not reveal an obvious phenotype in fat body cells following Sir2 over-expression, regardless of developmental stage or sex.

Figure 5. Sir2 overexpression in the fat body did not affect fat storage and fat cell morphology. Relative body fat content as quantified by coupled colorimetric assay (A) and body weight (B) of male and female UAS-Sir2>S₁106-Gal4 flies feeding on medium with or without 400 μM RU486 showed no significant differences. Diameters of fat droplets were measured in fat bodies of UAS-Sir2>S₁106-Gal4 L3 larvae after Bodipy staining (C) and no differences were observed. Bodipy staining furthermore reveals no differences in size and structure of fat cells as well as droplet size in L3 larvae (D, G), adult females (E, H) or males (F, I) after feeding on ethanol (top row) or RU486 (bottom row). A-C, Data are represented as means + SEM. Mann-Whitney-U-Test was performed using GraphPad Prism 6.00, n = 4 for coupled colorimetric assay and body weight, n = 7 for fat droplet diameter measurements. D-I, scale bar represents 50 μm.

Fly Strains and Husbandry

Males of the driver line S₁106-Gal4 (gift from R. Kuehnlein, MPI Goettingen, Germany [20]) were crossed to virgin females of UAS-Sir2 (gift from Kyung-Tai Min,[28]) to generate flies overexpressing Sir2 in the fat body after induction by RU486. As a control for RU486-dependent effects on lifespan a control crossing using the driver line and the w¹¹¹⁸ strain (Bloomington Drosophila Stock Center, # 5905) was performed. Fly stocks were raised on standard cornmeal-agar medium at 25 °C under a 12 h/12 h light/dark cycle.

Lifespan Experiments

For lifespan experiments the F1 generation of the crossings mentioned above were allowed to mate for two days after eclosion before they were set on medium containing 5 % (w/v) yeast extract (Becton Dickinson), 5 % (w/v) sucrose, 8.6 % cornmeal, 0.5 % (w/v) agar, 0.03 % (v/v) propionic acid, 0.3 % (v/v) methyl-4-hydroxybenzoat and either 400 μM RU486 (diluted from a 10 mM stock in 90 % ethanol) for the experimental or the corresponding volume of 90 % ethanol for the control groups. Flies were kept at a density of 25 animals per vial (68 ml, Greiner Bio One) at 25 °C, 40-60 % humidity and a 12 h/12h light/dark cycle. Medium was changed every 3 – 4 days and dead flies were counted.

Dietary Restriction (DR)

The DR experiments were performed with medium prepared as recommended in Ja et al. to provide sufficient water supply [23]. Control medium contained 5 % (w/v) yeast extract (Becton Dickinson), 5 % (w/v) sucrose, 8.6 % cornmeal, 0.5 % (w/v) agar, 0.03 % (v/v) propionic acid, 0.3 % (v/v) methyl-4-hydroxybenzoat. DR medium contained only 0.25 % yeast extract. w¹¹¹⁸ flies were allowed to mate for 2 days before genders were separated and either used for lifespan studies as described above or fed on control or DR medium for 7 d prior to dissection of fat bodies and RNA isolation.

Microarray Analysis

Microarray analyses were essentially performed as described earlier [41, 42]. In brief, cDNA synthesis from RNA isolated from fat bodies was performed with Prime Script RT (Takara) using the following primers: OdT T7 I (5'-GAG AGA GGA TCC AAG TAC TAA TAC GAC TCA CTA TAG GGA GAT TTT TTT TTT TTT TTT TTT TTT T G/A/C-3') and CapFinder Sp6rG (5'-CAG CGG CCG CAG ATT TAG GTG ACA CTA TAG A rGrGrG-3'). cDNA was amplified with OdT T7 II (5'-GAG AGA GGA TCC AAG TAC TAA TAC GAC TCA CTA TAG G-3') and Adaptor Sp6rG (5'-GAC GCC TGC AGG CGA TGA ATT TAG G-3') and LA Taq polymerase [43]. cDNA was transcribed with MEGAscript® T7 (Ambion) including aminoallyl-UTP (Ambion) and subsequently labeled with Alexa Fluor 555 or 647 (Invitrogen) for control or experimental sample, respectively. Samples were hybridized to Drosophila 14 k V2 (Canadian Drosophila Microarray Centre, Toronto, Canada) and scanned in a GenePix 4000B Microarray Scanner (Axon Instruments, Molecular Devices). Data acquisition, normalization and analysis including hierarchical clustering were carried out with the corresponding programs GenePix 6.0 and Acuity 4.1 (Axon Instruments, Molecular Devices). Scaled Venn diagrams were drawn with Wolfram Mathematica [44]. The microarray data have been deposited at the GEO database (Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/)) with the accession number GSE45974.

Quantitative real-time PCR

Mated Sir2>S₁106-Gal4 flies fed on medium containing RU486 or ethanol as described for lifespan experiments for 24 h. RNA was isolated from the abdomen of 3 animals for one biological replicate and reverse transcription was performed using OdT-T7I Primer and SuperScript III Reverse Transcriptase (Invitrogen). qPCR was performed in a StepOne Real-Time PCR System (Applied Biosystems) using the DyNAmo Flash SYBR Green qPCR Kit (Finnzymes) and the following primers: Sir2 forward (5'-GCC TCC AGG ACA GTT AGC AG-3'), Sir2 reverse (5'- CGT GAT ACC GGG AAT TGA AG-3'), RpL32 forward (5'- TTG GCT TCG GTT TCC GGC AAG-3'), RpL32 reverse (5'-ATC GAT CCG ACT GGT GGC GGAT-3'). RpL32 was used as a reference gene and expression data were analyzed using the method described by Pfaffl [45].

Relative Body Fat Quantification and Body Weight Measurement

A coupled colorimetric assay was performed using the Triglycerides Kit from Thermo Fisher Scientific (# 981786) according to the protocol from Hildebrandt et al. [27]. Briefly, after mating and feeding on medium containing RU486 or ethanol for 7 d, for one biological replicate 8 males or 5 females were weighed on a digital accuracy balance and homogenized in 1 ml 0.05 % Tween-20 with 3 zirconium beads (2,8 mm) for 2 min at 3.25 m/s in a Bead Ruptor 24 (Omni International). Lysates were heat-inactivated for 5 min at 70 °C, centrifuged and supernatant was incubated with the Triglyceride Solution for 30 min at 37 °C. Absorbance was measured at 562 nm and glyceryl trioleate served as a standard.

BODIPY Staining and Determination of Diameters of Lipid Droplets

After feeding on RU486 or control medium for 24 h L3 larvae or adults generated by crossing UAS-Sir2 flies to the driver line S1106-Gal4 were dissected, fixed and fat bodies were stained with a solution containing 1 μg/ml BODIPY (Invitrogen) and 5 μg/ml Hoechst (Sigma) for 30 min at room temperature. Slides were mounted in Mowiol/DABCO (Roth) and photographed with a Zeiss Axio imager 7.1 microscope equipped with a Zeiss Axiocam MRM camera within the next two days. 14 photographs taken from 7 biological replicates were analyzed per group with AxioVision 4.1 (Zeiss). To measure fat droplet diameters a line was drawn across each picture and only droplets intersecting with the line were measured using a measurement tool implemented in the program.

Starvation Resistance

Mated Sir2>S₁106-Gal4 flies were kept on 1 % agar containing either 400 μM RU486 or ethanol and vials were changed every 2-3 days. Dead flies were scored 1-3 times a day.

Statistics and Bioinformatics

All statistics were performed using GraphPad Prism version 6.00 for Windows, GraphPad Software, La Jolla California USA, www.graphpad.com. Lifespan and starvation survival data were analyzed by log rank test and data from qPCR, body fat quantification, body weight and diameters of lipid droplets by Mann-Whitney-U Test. Statistical analyses of Venn-diagram data were performed with Fisher's exact test.