Sirt4 Is a Mitochondrial Regulator of Metabolism and Lifespan in Drosophila Melanogaster

Sirt4 is a mitochondrial regulator of metabolism and lifespan in Drosophila melanogaster

Jason G. Wooda, Bjoern Schwerb,1, Priyan C. Wickremesinghea, Davis A. Hartnetta, Lucas Burhenna, Meyrolin Garciaa, Michael Lia, Eric Verdinb,2, and Stephen L. Helfanda,3

aDepartment of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, RI 02912; and bGladstone Institute of Virology and Immunology, University of California, San Francisco, CA 94158

Edited by Nancy M. Bonini, University of Pennsylvania, Philadelphia, PA, and approved December 29, 2017 (received for review November 28, 2017) Sirtuins are an evolutionarily conserved family of NAD+-dependent response to fasting and starvation as well as longevity. Importantly, deacylases that control metabolism, stress response, genomic sta- we show that transgenic expression of a mitochondrial sirtuin can bility, and longevity. Here, we show the sole mitochondrial sirtuin extend organismal lifespan. in Drosophila melanogaster, Sirt4, regulates energy homeostasis and longevity. Sirt4 knockout flies have a short lifespan, with in- Results creased sensitivity to starvation and decreased fertility and activity. Drosophila Sirt4 Is a Mitochondrial Protein. To study mitochondrial In contrast, flies overexpressing Sirt4 either ubiquitously or specif- sirtuin function in the fly, we examined the sequences of the five ically in the fat body are long-lived. Despite rapid starvation, Sirt4 known Drosophila sirtuin orthologs and found that only one of knockout flies paradoxically maintain elevated levels of energy them, Sirt4, contains a predicted N-terminal mitochondrial tar- reserves, including lipids, glycogen, and trehalose, while fasting, geting sequence (Fig. S1A). Drosophila Sirt4 (hereafter referred suggesting an inability to properly catabolize stored energy. to as dSirt4) is most closely related to the mouse SIRT4 protein Metabolomic analysis indicates several specific pathways are af- (45% identity; Fig. S1B). To test whether dSirt4 is localized to fected in Sirt4 knockout flies, including glycolysis, branched-chain mitochondria in flies, we transfected a dSirt4::Flag construct into amino acid metabolism, and impaired catabolism of fatty acids with S2 cells and performed immunoblot analysis of subcellular fractions.

chain length C18 or greater. Together, these phenotypes point to a The dSirt4::Flag signal was strongly enriched in the mitochondrial GENETICS role for Sirt4 in mediating the organismal response to fasting, and fraction relative to other subcellular fractions (Fig. 1A). To further ensuring metabolic homeostasis and longevity. assess the subcellular localization of dSirt4, we expressed either Flag-tagged dSirt4 or GFP-tagged dSirt4 proteins in S2 cells and aging | metabolism | sirtuins | mitochondria | Sirt4 examined subcellular localization via either immunofluorescence (dSirt4::Flag) or direct fluorescence (dSirt4::GFP) analysis (Fig. 1B; + additional images are shown in Fig. S2). Using MitoTracker dye or irtuins are a family of highly conserved NAD -dependent immunostaining of the mitochondrial protein MnSOD, we observed protein deacylases with roles in regulating many cellular S a high degree of overlap of fluorescence signal in the mitochondria processes, including genomic stability, metabolism, and longevity of transfected cells for both dSirt4 constructs, indicating that dSirt4 (1). In mammals, of the seven sirtuin family members, three localizes to mitochondria. (SIRT3, SIRT4, and SIRT5) are localized within mitochondria, where they have wide-ranging and overlapping effects on numerous metabolic pathways, including fatty acid metabolism, Significance tricarboxylic acid (TCA) cycle, glycolysis, reactive oxygen species (ROS), oxidative phosphorylation, protein metabolism, and the Sirtuins are a class of proteins known to regulate aspects of urea cycle (reviewed in ref. 2). Although mitochondrial sirtuins genomic stability, metabolism, and lifespan in many organisms. have a range of enzymatic activities and targets, an emerging In this study, we show that the mitochondrial sirtuin Sirt4 plays view suggests they work coordinately to regulate metabolic net- an important role in regulating the organismal response to works in mitochondria in response to changing environmental fasting as well as ensuring normal lifespan in Drosophila. Flies and nutrient conditions. SIRT3 displays robust deacetylase activity, lacking Sirt4 are short-lived, while flies overexpressing Sirt4 are and functions to clear ROS as well as activate fatty acid oxidation long-lived. Flies lacking Sirt4 display a number of metabolic (FAO) in response to fasting (3–7). SIRT5 has desuccinylase, defects, including sensitivity to starvation; decreased fertility demalonylase, and deglutarylase activities and up-regulates en- and activity; and an inability to utilize energy stores, particu- zymes in the urea cycle during fasting (2, 8). The targets and larly long-chain fatty acids, suggesting Sirt4 is important for enzymatic activity of SIRT4 remain enigmatic relative to the rest maintaining metabolic homeostasis. Our results suggest that of the sirtuins. Reported enzymatic activities for SIRT4 include boosting mitochondrial sirtuin activity may be an important both ADP ribosylation (9, 10) as well as a number of deacylase avenue for treating age-related metabolic decline and pre- activities, including removal of acetyl (11), lipoyl (12), glutaryl, serving healthy lifespan. methylglutaryl, and hydroxymethylglutaryl (13) adducts from ly- Author contributions: J.G.W., B.S., E.V., and S.L.H. designed research; J.G.W., B.S., P.C.W., sine residues. Likewise, metabolic targets of SIRT4 activity are D.A.H., L.B., M.G., and M.L. performed research; J.G.W. and B.S. analyzed data; and wide-ranging, including reported roles in insulin signaling, lipid J.G.W., B.S., and S.L.H. wrote the paper. metabolism, TCA cycle, pyruvate metabolism, and amino acid The authors declare no conflict of interest. – oxidation (9 13). The Drosophila melanogaster genome contains This article is a PNAS Direct Submission. five sirtuins named Sirt1, Sirt2, Sirt4, Sirt6, and Sirt7 after their Published under the PNAS license. closest mammalian orthologs. Of these five sirtuins, only one, Sirt4, 1Present addresses: Department of Neurological Surgery and Eli and Edythe Broad Center contains a predicted mitochondrial targeting sequence, suggesting of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, that Sirt4 may act as a more general mitochondrial sirtuin in this CA 94158. organism and perform functions distributed across other mito- 2Present address: Buck Institute for Research on Aging, Novato, CA 94945. chondrial sirtuins in mammals. Here, we report a genetic charac- 3To whom correspondence should be addressed. Email: [email protected]. terization of the Drosophila sirtuin Sirt4, and show it localizes to This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the mitochondria and plays a role in regulating the metabolic 1073/pnas.1720673115/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1720673115 PNAS Latest Articles | 1of6 Downloaded by guest on October 2, 2021 AB dSirt4 Mediates Response to Fasting and Starvation. In mammalian cells, mitochondrial sirtuins have been implicated in regulation of metabolic homeostasis via several different mitochondrial pathways (6–14, 16). To assess the importance of dSirt4 in maintaining normal metabolism during fasting and starvation, we performed a starvation sensitivity assay in dSirt4 knockout or dSirt4-overexpressing flies. We found that flies lacking dSirt4 were sensitive to starvation, and died much sooner than the genetically matched wild-type cohort (Fig. 3A and Fig. S5A). Conversely, we found that flies overexpressing dSirt4 were resistant to starvation, and able to survive longer than genetically Fig. 1. dSirt4 is localized to mitochondria. (A) Subcellular fractionation of matched uninduced controls in the absence of food (Fig. 3B and dSirt4::Flag cells shows mitochondrial localization. Homogenates from S2 cells expressing dSirt4::Flag were fractionated into mitochondria-enriched Fig. S5B). To complement the whole-organism starvation assays, heavy membrane (HM), light membrane (LM), and cytosolic (Cyt) fractions, we next examined whether dSirt4 expression was induced under and analyzed by Western blot. The mitochondrial protein MnSOD and cy- fasting or starvation conditions in wild-type flies. In mammals, tosolic proteins tubulin and Hsp90α are shown as fractionation controls. expression of both SIRT1 and SIRT3 is induced upon fasting (B) Immunofluorescence of dSirt4 constructs confirms mitochondrial localization. (18). Similarly, we observed a transcriptional up-regulation of dSirt4::Flag (Top) or dSirt4::GFP (Bottom) colocalizes with MitoTracker dye (Top) dSirt4 upon overnight fasting in the fat body of wild-type flies, μ or MnSOD (Bottom) in mitochondria of S2 cells. (Scale bar, 10 m.) indicating activation of dSirt4 under these conditions (Fig. 3C). Together, these results suggest that dSirt4 is responsive to nu- Modulating dSirt4 Levels Influences Organismal Lifespan. To facili- tritional inputs, and that functional dSirt4 in the fly is important tate physiological studies and examine the function of dSirt4 in a for mediating metabolic changes coincident with a fasting or whole-organism context, we used an available dSirt4 knockout starved state in the animal. line, and additionally generated fly lines overexpressing dSirt4. + For dSirt4 knockout flies, we used the Sirt4white 1 allele, in which the coding sequence of dSirt4 has been completely removed by homologous recombination, and backcrossed this line 20 times A 1 1 1118 into a w control line to generate genetically matched control ♀ ♂ and knockout lines. Additionally, to examine the consequences 0.5 0.5 of increasing dSirt4 expression, we generated transgenic flies 1118 containing a UAS-dSirt4 construct and crossed them with GAL4- w control w1118 control Survivorship dSirt4 KO dSirt4 KO based driver lines to increase expression of dSirt4. The dSirt4 0 0 knockout flies appeared phenotypically normal and healthy. 020406080 0 20406080100 B 1 1 Examination of overall mitochondrial function from dSirt4 knockout flies showed that respiratory function of extracted ♀ ♂ mitochondria (Fig. S3 A and B) and ATP levels in both whole 0.5 0.5 flies (Fig. S3C) and eviscerated abdomens (Fig. S3D) were in- da > w1118 da > w1118 distinguishable from wild-type controls. Survivorship da > UAS-dSirt4 da > UAS-dSirt4 0 0 Sirtuins are known to affect both metabolism and lifespan in 0204060801000 20406080100 several different species. However, to date, the role of mito- C 1 1 chondrial sirtuins in regulating organismal lifespan has not been reported. We thus asked whether genetically manipulating ♀ ♂ dSirt4 expression could affect organismal lifespan. Compared 0.5 0.5 with a genetically matched wild-type control cohort, flies lack- tub > w1118 tub > w1118 ing dSirt4 were substantially shorter lived (Fig. 2A). However, Survivorship tub > UAS-dSirt4 tub > UAS-dSirt4 0 0 despite a notably shorter lifespan than controls, dSirt4 knockout 0204060801000 20406080100 flies respond to dietary restriction with the normally expected D 1 1 increase in lifespan (Fig. S4A) and physical activity (Fig. 3D and Fig. S5 C–E), suggesting these flies are generally healthy and ♀ ♂ not grossly impaired. 0.5 0.5 Importantly, transgenic flies overexpressing dSirt4 exhibit an ppl > w1118 ppl > w1118 Survivorship ppl > UAS-dSirt4 ppl > UAS-dSirt4 extended lifespan compared with genetically matched controls 0 0 when driven by the ubiquitously expressing driver da-GAL4 (Fig. 0 20406080100020406080 2B)ortub-GAL4 (Fig. 2C; female flies only). Based on studies of Age (days) Age (days) sirtuin functions in mammalian liver and their reported roles in Fig. 2. Effect of dSirt4 on lifespan. (A) dSirt4 knockout (KO) flies are short- regulating lipid metabolism (6, 7, 11, 12, 14–16), we next tested lived compared with genetically matched w1118 controls (28% median life- whether specific expression of dSirt4 in the fat body was suffi- span decrease in females, 17% median lifespan decrease in males). (B) Flies cient to extend lifespan. The fat body is the primary metabolic overexpressing dSirt4 ubiquitously (da-GAL4 > UAS-dSirt4) are long-lived regulatory organ of the fly, serving the metabolic functions of the compared with da-GAL4 > w1118 controls (20% median lifespan increase in mammalian liver and adipocytes (17). When driven by the fat females and males). (C) Female flies overexpressing dSirt4 ubiquitously (tub- 1118 body-specific ppl-GAL4 driver, transgenic flies overexpressing GAL4 > UAS-dSirt4) are long-lived compared with tub-GAL4 > w controls dSirt4 only in the fat body were long-lived compared with ge- (13% median lifespan increase in females, no significant median lifespan increase in males). (D) Flies overexpressing dSirt4 specifically in the fat body netically matched controls (Fig. 2D). The magnitude of the 1118 (ppl-GAL4 > UAS-dSirt4) are long-lived compared with ppl-GAL4 > w lifespan extension with the fat body-specific ppl-GAL4 driver was controls (14% median lifespan increase in females, 20% median lifespan similar to that observed with the ubiquitously expressing drivers increase in males). Log-rank P < 10−10 for all lifespans shown, except tub- (Fig. 2D). These findings demonstrate that increasing dSirt4 ex- GAL4 > UAS-dSirt4 males (shown in C, Right), which are not significant. Full pression can extend the lifespan in the fly, and that this effect statistics, including n (number of individuals assayed), median, mean, and may be primarily mediated through dSirt4 actions in the fat body. maximum lifespan values for all experiments, are presented in Table S1.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1720673115 Wood et al. Downloaded by guest on October 2, 2021 1118 1118 dSirt4 Knockout Flies Exhibit Impaired Ability to Use Energy Stores ABw control da-GAL4 > w C 2.5 dSirt4 KO da-GAL4 > UAS-dSirt4 During Fasting. Because dSirt4 knockout flies starved more rapidly 1 1 2 than control flies, we sought to better understand how these 1.5 1 animals stored and processed energy when fasting. We assayed 0.5 0.5 the levels of basic energy storage metabolites, including lipids, 0.5 glycogen, and carbohydrates, over a 24-h time course in flies as Fraction alive 0 0 0 0 122436486072 0 122436486072 they fasted (Fig. 4). Sensitivity to starvation could be caused by Hours Hours dSirt4 Relative levels Fed differences in total energy stores; rates of synthesis, mobilization, DEFasted 80 1118 1118 w control HC or catabolism of energy stores; or a combination of these factors. w control 8 250 dSirt4 KO HC As in mammals, triglycerides (TAGs) are the primary form of 60 dSirt4 KO w1118 control LC 6 200 dSirt4 KO LC energy storage in the fly, and are rapidly oxidized in the mito- 40 150 chondria to provide energy during fasting. In the continuously thousands) ( 4 fed state, dSirt4 knockout flies have moderately higher levels of ts 100 20 2 total TAGs than genetically matched control flies (Fig. 4A). In

oun 50 Counts / fly 30 m

C wild-type flies, TAG levels drop consistently over the course of 0 0 0 fasting, such that by 24 h, flies maintain only 22% of initial TAG 0 12 24 36 48 60 72 tal 510152025 o Cumulative eggs laid / ﬂy Time (hours) T LightDark Days reserves. Strikingly, dSirt4 knockout flies show a much slower loss of TAG levels during fasting, retaining 60% of their TAG stores Fig. 3. Effects of dSirt4 on starvation, activity, and fertility. (A) dSirt4 after 24 h (Fig. 4A). We next measured levels of glycogen, an knockout (KO) flies are starvation-sensitive relative to genetically w1118 1118 = = = energy storage molecule that is hydrolyzed during fasting to yield matched controls (w : median survival 41 h, mean 40.9 h, n 96; carbohydrates for energy. Wild-type control and dSirt4 knockout dSirt4 KO: median survival = 32 h, mean = 34.1 h, n = 97). Male flies are − shown, log-rank P < 10 10.(B) Flies overexpressing dSirt4 ubiquitously (da- flies contained indistinguishable levels of glycogen in the fed GAL4 > UAS-dSirt4) are starvation-resistant relative to da-GAL4 > w1118 state (Fig. 4B). Similar to our observations with TAG, during controls (control: median survival = 43 h, mean = 43.1 h, n = 100; dSirt4 fasting, wild-type control flies lost glycogen more quickly than transgenic: median survival = 49 h, mean = 48.8 h, n = 99). Male are flies dSirt4 knockouts, with wild-type controls maintaining only 15% − shown, log-rank P < 10 10.(C) dSirt4 transcript levels increase upon over- of fed-state glycogen levels after 24 h and dSirt4 knockouts night (16 h) fasting in fat bodies of 10-d-old wild-type female flies. Data maintaining 33% of fed-state glycogen levels (Fig. 4B). < represent mean of three biological replicates, and error bars are SEM (P Finally, we examined the levels during fasting of glucose and GENETICS 0.01, unpaired two-tailed t test on delta threshold cycle values). (D, Left) dSirt4 trehalose, the major circulating sugars in Drosophila. Trehalose KO flies have decreased spontaneous activity compared with controls over a levels in fed flies were indistinguishable between dSirt4 knock- 3-d period. Gray shading indicates the dark period (12-h cycle). (D, Right) Bar outs and wild-type controls (Fig. 4C). Wild-type control flies graph displays total, light period, and dark period counts shown in the activity showed relatively stable trehalose levels during early stages of plot. Data represent the average of three replicate vials, with 20 female flies fasting, with total stores declining to 50% by 24 h. The dSirt4 per vial on high-calorie (15% SY) food, and are presented as the number of knockout flies, by contrast, showed a 33% increase in trehalose counts per 30-min bin per fly. (E) dSirt4 KO flies exhibit impaired fertility levels by 8 h of fasting, and levels remained consistently high relative to controls. Cumulative eggs laid are shown for both genotypes on both high-calorie (HC: 15% SY) and low-calorie (LC: 5% SY) diets. n = 10 vials, thereafter throughout the time course (Fig. 4C). Glucose levels five flies per vial. Error bars represent SEM (P < 0.01, unpaired two-tailed were 22% higher in dSirt4 knockout flies than controls in the fed t test) between genotypes at all time points for both diets. state (Fig. 4D). Upon fasting, levels remained relatively stable in

Changes in food availability or energy status are usually TAG Glycogen reflected in physical activity. We measured the spontaneous ac- w1118 control AB4 * *** tivity of flies lacking dSirt4 over a multiday period using an au- dSirt4 KO n.s. 0.6 tomated activity monitor. We observed that dSirt4 knockout flies 3 *** ** – *** had an overall lower activity level than controls (35 70% de- *** crease depending on condition), and that this difference was 2 0.4 particularly pronounced during the normally more active day- ** AG] / [Protein] 1 0.2 time hours (Fig. 3D and Fig. S5 C–E). Although dSirt4 knockout [T flies exhibited overall lower physical activity, they maintained 0 0 [Glycogen] / [Protein] normal circadian periodicity of movement, with peaks in activity 0 8 16 24 0 8 16 24 surrounding the light/dark transitions (Fig. 3D and Fig. S5 C–E). Hours fasted Hours fasted Finally, it is known that Drosophila fertility is correlated with CDTrehalose Glucose nutrient availability (19). We assayed fertility of control and ** ** ** * ** dSirt4 knockout flies by measuring the total number of eggs laid 0.3 n.s. 0.6 * * daily by a cohort of females over the first 3 wk of life. Flies lacking dSirt4 displayed decreased fertility, with 33% fewer eggs 0.2 0.4 produced than wild-type controls on both high- and low-calorie content food (Fig. 3E). This suggests that dSirt4-deficient flies 0.1 0.2 are unable to properly direct energy toward egg production. 0 [Trehalose] / [Protein] [Trehalose] 0 Lifespan and fertility are frequently inversely related, as flies 0 8 16 24 [Glucose] / [Protein] 0 8 16 24 with decreased fertility, such as ovaryless flies, typically exhibit a Hours fasted Hours fasted long lifespan (20). However, dSirt4 knockout flies are short-lived despite exhibiting lower fertility, indicating a severe effect of Fig. 4. Levels of energy storage metabolites in dSirt4 knockout (KO) flies dSirt4 loss on normal metabolic homeostasis. Conversely, we during fasting. (A) TAG levels in control (black) and dSirt4 KO (red) male flies, assayed every 8 h over a 24-h fasting period. Corresponding mea- observed that flies ubiquitously overexpressing dSirt4 had no surements for glycogen (B), trehalose (C), and glucose (D) under the same significant difference in fertility from controls, despite having conditions are shown. Data represent the mean of five biological replicates, longer lifespans (Fig. S4B). This indicates that the effects of with five flies per replicate. All metabolite measurements were normalized dSirt4 on lifespan are not mediated by changes in fertility. To- against total protein in each sample and reported in units of milligrams per gether, the starvation, activity, and fertility phenotypes point milliliter. Error bars represent SEM. * = 0.01 < P < 0.05; ** = 0.001 < P < 0.01; toward a defect in metabolic regulation in dSirt4-deficient flies. *** = P < 0.001 (unpaired two-tailed t test). n.s., not significant.

Wood et al. PNAS Latest Articles | 3of6 Downloaded by guest on October 2, 2021 ACGlycolysis B BCAA 2 TCA w1118 control fed 3 3 dSirt4 KO fed 1.5 w1118 control fasted dSirt4 KO fasted 2 2 1

1 1 0.5 Relative quantitation

0 0 0

DHAP ribose valine leucine lactic acid isoleucine citric acid malic acid pyruvic acid isocitric acidsuccinic acidfumaric acid

phosphoenolpyruvateD-ribose-5-phosphate glucose-1-phosphateglucose-6-phosphate D 2.5 E Free fatty acids Fatty acid methyl esters (FAME) 3 2

1.5 2

1 1 Relative quantitation 0.5

0 0

laurate C12:0 lauric acid C12:0 myristate C14:0 palmitate C16:0stearate oleate C18:0 C18:1n-9 behenate C22:0 myristic acidpalmitic C14:0 acidstearic C16:0 acid C18:0oleic acid C18:1n-9 linoleatearachidate C18:2n-6 C20:0 lignocerate C24:0 linoleic acid C18:2n-6 myristoleate C14:1 pentadecanoatepalmitoleate C15:0 C16:1n-7linolelaidate C18:2n-6 docosadienoate C22:2n-6 docosahexaenoate C22:6n-3

Fig. 5. Comparative metabolomic profiling of male dSirt4 knockout (KO; red/pink) and w1118 control (black/gray) flies in both fed (black/red bars) and fasted (gray/pink bars) states. Relative levels of glycolytic metabolites (A), BCAAs (B), citric acid cycle metabolites (C), and free fatty acids (D) are shown for dSirt4 KO and control flies. Measurements were performed using a small metabolite GC-MS protocol. (E) Relative levels of fatty acids in total lipid profile as determined by FAME GC-MS. Due to the normalization procedure, the FAME analysis does not measure total TAG levels in each sample but, instead, measures the relative abundance of different fatty acid species within the total lipid pool of each sample. Levels of each metabolite were normalized to the control fed condition (black bar). Bars represent the mean of six replicates, and error bars represent SEM. The P values of significance tests of presented data are given in Table S2.

both genotypes for the first 16 h, with wild-type and dSirt4 glucose-1-phosphate and glucose-6-phosphate are substrates for knockout levels eventually declining to 62% and 79%, re- trehalose synthesis, in addition to being used for glycolysis (Fig. spectively, of fed-state levels at 24 h (Fig. 4D). Taken together, 4C). In addition to lipids, branched-chain amino acids (BCAAs) these metabolic assays demonstrate that dSirt4 knockout flies can be oxidized in the mitochondria to provide fuel during starve more rapidly than controls despite having equivalent or fasting, and mammalian SIRT4 has been implicated in regulation higher levels of energy reserves than wild-type controls in the fed of this pathway (13). Consistent with this reported role of SIRT4, state. Furthermore, dSirt4 knockout flies consistently maintain BCAA levels were substantially higher in dSirt4 knockouts than higher levels of energy storage metabolites during fasting, in- controls, in both fed and fasted states (Fig. 5B). Krebs/citric acid dicating that they may suffer from an inability to properly ca- cycle metabolites were generally comparable between dSirt4 tabolize energy reserves during periods of fasting. These data knockouts and controls, although citrate, fumarate, and malate point toward a role for dSirt4 in regulating the organismal levels were significantly lower in dSirt4 knockouts in the fasted metabolic response to fasting and starvation. state (Fig. 5C). Examination of lipids showed that dSirt4 knockouts had slight increases in C12, C14, and C16 saturated free fatty acids dSirt4 Knockout Flies Show Altered Metabolite Profiles. To further compared with controls in the fed state, but no significant differ- investigate the specific metabolic differences between dSirt4 ences in the fasted state (Fig. 5D). However, levels of the un- knockout and wild-type control flies, we performed a compara- saturated free fatty acids linoleic acid (C18:2n-9) and oleic acid tive metabolomics analysis that examined metabolite levels in a (C18:1n-6) were higher in dSirt4 knockouts than controls in both number of different pathways, in both fed and fasted states (Fig. the fed and fasted states, with the difference much greater in the 5). We found that dSirt4 knockout flies exhibited a number of fasted state (Fig. 5D). differences from control flies in important metabolic pathways. We next sought to more precisely assay the relative composi- In the fed state, dSirt4 knockout flies had substantially higher tion of lipids in dSirt4 knockout and control flies during fasting. levels of many glycolytic intermediates compared with control To do this, we performed an alternate comparative metabolomic flies (Fig. 5A). In the fasted state, most glycolytic metabolites analysis using a fatty acid methyl ester (FAME)-based method to were not substantially higher than in controls, and glucose-1- examine the relative abundance of different fatty acids within the phosphate and glucose-6-phosphate levels were significantly total lipid pool of each sample. The dSirt4 knockout flies showed lower. This can perhaps be explained by the substantially higher a number of differences from controls in fatty acid profiles for fasting levels of trehalose observed in dSirt4 knockout flies, as both saturated and unsaturated fatty acids (Fig. 5E). In the fed

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1720673115 Wood et al. Downloaded by guest on October 2, 2021 state, dSirt4 knockouts showed modestly lower levels of shorter role for SIRT3 has been described in mammals, where it deacety- chain fatty acids (C12:0, C14:0, and C14:1), elevated levels of lates and activates both long-chain acyl-CoA dehydrogenase and palmitate (C16:0) and palmitoleic acid (C16:1), and no signifi- VLCAD to stimulate FAO in response to fasting (6, 7, 16). cant differences in longer chain fatty acids (Fig. 5E), compared The complexity of metabolic networks and homeostatic feed- with controls. However, in the fasted state, dSirt4 knockouts back mechanisms suggests dSirt4 may exert its effects through showed a strikingly different fatty acid profile from controls, with one or more of a number of different mechanisms. In addition to much lower levels of C12 and C14 fatty acids, unchanged fatty acids, our metabolomics results suggest dSirt4 may be in- C16 levels, and significantly higher levels of long-chain and very- volved in regulating both glycolysis, perhaps through effects long-chain fatty acids in the C18–C24 range (Fig. 5E). Analyzed on pyruvate dehydrogenase and/or pyruvate carboxylase, and together with the total TAG time course assay (Fig. 4A), which BCAA oxidation, possibly through methylcrotonyl-CoA carbox- measures absolute lipid levels, these data show that wild-type ylase 1 (MCCC1) and/or short/branched-chain–specific acyl-CoA flies deplete fat stores during fasting, and do so relatively dehydrogenase (ACADSB). Regulating the activity of any or all evenly across the spectrum of fatty acids. The dSirt4 knockout of these pathways could result in our observed phenotypes, in- flies, however, maintain overall higher levels of TAG during cluding reduced longevity and sensitivity to starvation despite fasting, and specifically retain lipids with chain lengths of C18 or abundant energy stores. Interestingly, mammalian mitochondrial greater. This suggests a critical role for dSirt4 in regulating me- sirtuins have also been directly implicated in regulating all of tabolism of these long-chain and very-long-chain fatty acids. these processes (2). Activity of dSirt4 on a broader network of targets and pathways would be consistent with recent reports in Discussion mammals describing complex and wide-ranging mitochondrial Sirtuins have been implicated in a wide variety of cellular processes sirtuin target networks (25–29). and age-related disease states, and have emerged as important Upon prolonged fasting, a number of different metabolic regulators of genomic stability, metabolism, and longevity (1, 2). In programs are activated to ensure organismal survival, including this study, we show that dSirt4 is a mitochondrial protein, and we glycogenolysis, gluconeogenesis, ketosis, lipolysis and FAO, auto- used the D. melanogaster model organism to examine the whole- phagy, and proteolysis. Similar to mammalian SIRT3, we observed organism physiological effects of genetically manipulating mito- that dSirt4 expression is up-regulated in the fat body of the fly in chondrial sirtuin expression. Class II (SIRT4) and class III (SIRT5) response to fasting. Multiple mammalian studies point to a consis- sirtuins are evolutionarily the most ancient of the sirtuin family, tent role for mitochondrial sirtuins in regulating switching between

and Drosophila lacks a SIRT5 homolog, leaving dSirt4 as the sole fuel sources in the mitochondria in response to fasting and caloric/ GENETICS predicted mitochondrial sirtuin in this organism (21) (Fig. S1A). dietary restriction (6–8, 11–13, 30). Despite not knowing the enzy- Mammalian mitochondrial sirtuins have overlapping and some- matic specificity of dSirt4, the physiological phenotypes reported in times opposing regulatory effects on cellular processes. For ex- this study are consistent with a similar role for dSirt4 acting within ample, SIRT3 and SIRT4 show opposite effects on FAO as well as the mitochondria to regulate one or more metabolic pathways and adiposity (6, 11, 15). The spectrum of mitochondrial sirtuin activity facilitate fuel type switching in response to environmental inputs, and targets in Drosophila is unknown; however, given the lack such as food availability. These processes appear to be increasingly of other clear mitochondrial sirtuin orthologs, it is likely that important with advancing age, and maintaining dSirt4 function is dSirt4 may have evolved in flies to perform functions that are di- crucial for normal lifespan, as dSirt4 knockouts live much shorter vided between multiple mitochondrial sirtuins in other organisms. than controls. Notably, flies overexpressing dSirt4 show lifespan Consistent with this idea, several phenotypes in our dSirt4 knockout extension without a decline in fertility, indicating that boosting flies, including transcriptional induction by fasting and defects in mitochondrial sirtuin activity may have beneficial effects on both + lipid utilization, are reminiscent of SIRT3 functions in mammals. longevity and metabolism. The requirement of NAD as a cofactor We report that dSirt4 knockout flies display a number of for sirtuin activity links these enzymes to the energetic landscape of phenotypes consistent with an inability to properly process and the cell, and positions them as ideal environmental energy sensors. + use energy stores. They are sensitive to starvation, a state that NAD levels are known to decline with aging, and there is emerging + requires a major metabolic shift away from dietary energy and evidence that supplementation with NAD , which can boost sirtuin toward stored energy reserves to maintain survival. Additionally, activity, can delay, and may possibly reverse, some of the deleterious dSirt4 knockout flies exhibit a markedly shorter lifespan than effects of aging (31–33). Therefore, as organisms age, it is likely that their wild-type controls. These phenotypes are likely related, as they have difficulty maintaining adequate dSirt4 function in the face both metabolic efficiency and feeding rate decline substantially of declining metabolic homeostasis, leaving them unable to properly – with age (22 24), suggesting flies become more dependent on process and utilize energy stores. This model would explain our stored energy and less dependent on dietary energy as they age. observation that dSirt4 overexpression extends lifespan, as increased The dSirt4 knockout females produce fewer eggs, a process that gene dosage may be sufficient to counter the age-related loss of is very energy-intensive and requires large amounts of lipids to function of dSirt4 and forestall the metabolic and longevity conse- form mature oocytes. Interestingly, despite exhibiting numerous quences of impaired dSirt4 activity. Furthermore, this suggests that phenotypes suggesting metabolic deficiency under nutrient activating mitochondrial sirtuin function may prove similarly useful stress, dSirt4 knockout flies in the fed state appear phenotypi- for improving metabolic function in mammals, particularly with age. cally normal and maintain normal or elevated levels of energy Impaired metabolic homeostasis is a hallmark of aging, and mito- reserves in the form of TAGs and glycogen. Additionally, their chondrial sirtuins are well situated to act as guardians of cellular mitochondrial respiratory function and ATP levels are identical and organismal metabolism, and to ameliorate the functional de- to those of controls. Although dSirt4 knockout flies starve more cline and disease states associated with aging. rapidly than control flies, they maintain higher levels of TAGs, glycogen, and trehalose while fasting. Furthermore, detailed Materials and Methods metabolomic analysis indicates that the fat reserves maintained Fly Stocks and Husbandry. Flies were maintained at 25 °C on a 12-h light/dark by these flies during fasting are highly enriched for long-chain cycle at 60% relative humidity. High-calorie food [15% sugar/yeast (SY)] was and very-long-chain fatty acids with chain lengths of C18 or 15% dextrose/15% yeast/2% agar, and low-calorie food (5% SY) was 5% dex- + greater. There are multiple acyl-CoA dehydrogenase activities trose/5% yeast/2% agar. Bloomington line 8840, containing the Sirt4white 1 ho- within the mitochondria, with preferences for substrates with dif- mologous recombination deletion allele, was backcrossed 20 times into a w1118 ferent chain lengths. This points to a potential role for dSirt4 in control to generate genetically matched control and Sirt4 knockout flies. The regulating oxidation of these longer chain fatty acids, perhaps by coding DNA sequence of full-length Drosophila Sirt4 (CG3187-RC, transcript regulating import of longer chain fatty acids into the mitochondria, variant C) was cloned into the pUASt-based pTW vector (Drosophila Geno- or regulating activity of the very-long-chain acyl-CoA dehydrogenase mics Resource Center) and injected into w1118 flies to generate UAS-Sirt4 (VLCAD) complex that catalyzes the first step in FAO. A similar transgenic flies (transgene insertion on chromosome 3), together with a

Wood et al. PNAS Latest Articles | 5of6 Downloaded by guest on October 2, 2021 genetically matched control. These flies were crossed to the indicated GAL4 Metabolite Assays. Plate assays for TAG, glycogen, trehalose, and glucose

drivers, and the resulting F1 flies were used for transgenic experiments. were performed as described by Tennessen et al. (35), using the Serum Tri- glyceride Determination Kit (Sigma TR0100) and Infinity Glucose Hexokinase Lifespans. Lifespan assays were performed by mating newly eclosed flies of Reagent (Thermo TR15421). Five 30-d-old male flies were used for each each experimental genotype for 3 d, and then separating males and females sample, with five biological replicates for each condition. Flies were fasted and seeding vials at 25 male or female flies per vial, with 10 vials per ge- on 2% agar as described above, and were collected and assayed at 0, 8, 16, notype/condition (n = 250 for each sex). Flies were passed to new food vials, and 24 h of starvation time to generate a time course. and dead flies were counted every other day for the length of the assay. Lifespans were performed on 5% SY food, except in the experiment shown Metabolomics. Metabolomic analysis was performed by the University of Utah > in Fig. 2D (ppl-GAL4 UAS-dSirt4), which was performed on 15% SY food. Metabolomics Core. Two metabolomic analyses were performed, each using Lifespan statistics were calculated using the OASIS2 online tool (34). Maxi- six replicates of 20 male flies per sample either continuously fed or fasted for mum lifespan was calculated as the mean lifespan of the longest surviving 24 h. The first, a small-metabolite GC-MS protocol, is described by Tennessen 10% of the cohort. All lifespan assays were repeated at least twice, and et al. (35), and these data are presented in Fig. 5 A–D. The second is a FAME representative experiments are shown in Fig. 2 and Fig. S4A. Full details of GC-MS protocol that measures fatty acid composition in the lipid fraction lifespan trials are presented in Table S1. specifically (Fig. 5E). For each replicate, 20 adult flies were homogenized in 2:1 chloroform/methanol (vol/vol) to extract lipids. Samples were washed in Starvation Assays. Flies were raised on 15% SY food for 10 d, sexed, and 0.9% NaCl, and the lower chloroform phase was dried under vacuum. The seeded at a density of 10 flies per vial, and then given at least 24 h to recover resulting lipid residue was derivitized to FAME by treatment with 12% BCl - from anesthesia. To synchronize feeding, flies were placed in 2% agar vials 3 methanol (wt/wt) and heating at 60 °C for 10 min. Following the addition of for 4 h to fast; they were then placed on 15% SY for 2 h to feed and transferred back to 2% agar vials to start the assay. Flies were monitored and equal volumes of hexane and water, the upper hexane layer was dried and counted every 2–6 h until all flies in the vial had died (n = 100 for each analyzed by GC-MS. The individual FAME peaks were identified by MS and condition). Starvation curve statistics were computing using OASIS2 (34). All quantified by peak area. starvation assays were repeated at least twice, and representative experi- Additional methods are described in SI Materials and Methods. ments are shown in Fig. 3 A and B and Fig. S5 A and B. ACKNOWLEDGMENTS. We thank Suzanne Hosier for technical assistance Activity Monitor. Ten-day-old flies were seeded at a density of 20 female or and Will Lightfoot for fly food preparation. We also thank the Bloomington male flies per vial with three replicate vials per condition, using the Dro- Drosophila Stock Center for fly stocks; James Cox and the University of Utah sophila Activity Monitoring (TriKinetics) system with constant monitoring Metabolomics core for MS services and analysis; and Robert Reenan, Nicola Neretti, John Sedivy, and Matt Hirschey and his laboratory members for over a period of 4 d. All counts from each monitor were summed and binned helpful discussions. The pAWG, pAWF, and pTW vectors were obtained from into 30-min bins, and replicates were averaged together and plotted. the Drosophila Genomics Resource Center (Indiana University), which is supported by NIH Grant 2P40OD010949. This work was supported by an Ellison/ Fertility. Five female and five male flies were placed into each of 10 vials of American Federation for Aging Research (AFAR) Postdoctoral Fellowship 15% SY food for each condition. Flies were passed to new food vials every Award and a Nathan Shock Center Pilot Grant (to J.G.W.) and by NIA Grants 24 h, and total eggs laid over each 24-h period were recorded daily for a AG16667 and AG24353, a Glenn/AFAR Breakthroughs in Gerontology period of 3 wk. Award, and NIH Program Project Grant AG51449 (to S.L.H.).

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