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. 2018 Feb 13;115(7):1564-1569.
doi: 10.1073/pnas.1720673115. Epub 2018 Jan 29.

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

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Free PMC article

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

Jason G Wood et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Sirtuins are an evolutionarily conserved family of NAD+-dependent deacylases that control metabolism, stress response, genomic stability, and longevity. Here, we show the sole mitochondrial sirtuin in Drosophila melanogaster, Sirt4, regulates energy homeostasis and longevity. Sirt4 knockout flies have a short lifespan, with increased sensitivity to starvation and decreased fertility and activity. In contrast, flies overexpressing Sirt4 either ubiquitously or specifically in the fat body are long-lived. Despite rapid starvation, Sirt4 knockout flies paradoxically maintain elevated levels of energy reserves, including lipids, glycogen, and trehalose, while fasting, suggesting an inability to properly catabolize stored energy. Metabolomic analysis indicates several specific pathways are affected in Sirt4 knockout flies, including glycolysis, branched-chain amino acid metabolism, and impaired catabolism of fatty acids with chain length C18 or greater. Together, these phenotypes point to a role for Sirt4 in mediating the organismal response to fasting, and ensuring metabolic homeostasis and longevity.

Keywords: Sirt4; aging; metabolism; mitochondria; sirtuins.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
dSirt4 is localized to mitochondria. (A) Subcellular fractionation of dSirt4::Flag cells shows mitochondrial localization. Homogenates from S2 cells expressing dSirt4::Flag were fractionated into mitochondria-enriched heavy membrane (HM), light membrane (LM), and cytosolic (Cyt) fractions, and analyzed by Western blot. The mitochondrial protein MnSOD and cytosolic proteins tubulin and Hsp90α are shown as fractionation controls. (B) Immunofluorescence of dSirt4 constructs confirms mitochondrial localization. dSirt4::Flag (Top) or dSirt4::GFP (Bottom) colocalizes with MitoTracker dye (Top) or MnSOD (Bottom) in mitochondria of S2 cells. (Scale bar, 10 μm.)
Fig. 2.
Fig. 2.
Effect of dSirt4 on lifespan. (A) dSirt4 knockout (KO) flies are short-lived compared with genetically matched w1118 controls (28% median lifespan decrease in females, 17% median lifespan decrease in males). (B) Flies overexpressing dSirt4 ubiquitously (da-GAL4 > UAS-dSirt4) are long-lived compared with da-GAL4 > w1118 controls (20% median lifespan increase in females and males). (C) Female flies overexpressing dSirt4 ubiquitously (tub-GAL4 > UAS-dSirt4) are long-lived compared with tub-GAL4 > w1118 controls (13% median lifespan increase in females, no significant median lifespan increase in males). (D) Flies overexpressing dSirt4 specifically in the fat body (ppl-GAL4 > UAS-dSirt4) are long-lived compared with ppl-GAL4 > w1118 controls (14% median lifespan increase in females, 20% median lifespan increase in males). Log-rank P < 10−10 for all lifespans shown, except tub-GAL4 > UAS-dSirt4 males (shown in C, Right), which are not significant. Full statistics, including n (number of individuals assayed), median, mean, and maximum lifespan values for all experiments, are presented in Table S1.
Fig. 3.
Fig. 3.
Effects of dSirt4 on starvation, activity, and fertility. (A) dSirt4 knockout (KO) flies are starvation-sensitive relative to genetically w1118 matched controls (w1118: median survival = 41 h, mean = 40.9 h, n = 96; 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-GAL4 > UAS-dSirt4) are starvation-resistant relative to da-GAL4 > w1118 controls (control: median survival = 43 h, mean = 43.1 h, n = 100; dSirt4 transgenic: median survival = 49 h, mean = 48.8 h, n = 99). Male are flies shown, log-rank P < 10−10. (C) dSirt4 transcript levels increase upon overnight (16 h) fasting in fat bodies of 10-d-old wild-type female flies. Data represent mean of three biological replicates, and error bars are SEM (P < 0.01, unpaired two-tailed t test on delta threshold cycle values). (D, Left) dSirt4 KO flies have decreased spontaneous activity compared with controls over a 3-d period. Gray shading indicates the dark period (12-h cycle). (D, Right) Bar graph displays total, light period, and dark period counts shown in the activity plot. Data represent the average of three replicate vials, with 20 female flies per vial on high-calorie (15% SY) food, and are presented as the number of counts per 30-min bin per fly. (E) dSirt4 KO flies exhibit impaired fertility 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, five flies per vial. Error bars represent SEM (P < 0.01, unpaired two-tailed t test) between genotypes at all time points for both diets.
Fig. 4.
Fig. 4.
Levels of energy storage metabolites in dSirt4 knockout (KO) flies 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 measurements for glycogen (B), trehalose (C), and glucose (D) under the same conditions are shown. Data represent the mean of five biological replicates, with five flies per replicate. All metabolite measurements were normalized against total protein in each sample and reported in units of milligrams per milliliter. Error bars represent SEM. * = 0.01 < P < 0.05; ** = 0.001 < P < 0.01; *** = P < 0.001 (unpaired two-tailed t test). n.s., not significant.
Fig. 5.
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.

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