Alpha-ketoglutarate extends Drosophila lifespan by inhibiting mTOR and activating AMPK

AKG extends Drosophila lifespan

First, we investigated the potential of dietary supplementation with AKG (from 0 to 1000 μM) to increase the lifespan of Drosophila (Supplementary Figure 1). Positive effects on lifespan were seen at intermediate doses of 1, 5, and 10 μM (Figure 1A); however, there were no effects below or above these concentrations, reflecting a typical nonmonotonic dose–response curve. The dose–response analysis showed that supplementation of flies’ diets with 5 μM AKG increased median lifespan by up to 8.54% (Figure 1A and 1B). In subsequent experiments, we selected 5 μM as the optimal dose. To assess whether the longevity benefits of AKG were indirectly caused by drug aversion and/or rejection that led to self-imposed dietary restriction (DR), we evaluated AKG’s effect on food intake. During a 1-hour feeding assay, AKG had no effect on food consumption (Figure 1C).

Figure 1. Effect of AKG on Drosophila lifespan. (A) Effects of low AKG concentrations on Drosophila lifespan. Treatment with low-dose AKG (0.5, 1, 5, and 10 μM) extended the lifespan of W^Dah females compared to control flies on 1x sugar/yeast/agar (SYA) food (1 μM vs 0 μM AKG, p < 0.001; 5 μM vs 0 μM AKG, p < 0.001; 10 μM vs 0 μM AKG, p = 0.002; log-rank test). (B) Effects of different concentrations of AKG on Drosophila median lifespan. (C) Food consumption measurement. There was no difference in food consumption between control and AKG-treated groups (ns = nonsignificant, p > 0.05, two-tailed Student’s t-tests).

Effects of AKG on fecundity, physical activity, and stress tolerance

To evaluate potential mechanisms by which AKG prolonged lifespan, we assessed whether AKG affected fecundity and physical activity of flies during aging. We found that diets supplemented with 1 and 5 μM AKG significantly decreased fly fecundity, while 10 μM AKG had no significant effect (Figure 2A).

Figure 2. Effects of AKG on reproduction, vertical climbing ability, and stress tolerance in w^Dah flies. (A) Fecundity assay. AKG decreased fecundity at concentrations of 1 and 5 μM (1 μM vs 0 μM AKG, p = 0.0013; 5 μM vs 0 μM AKG, p = 0.005; Wilcoxon rank test) and had no significant effect at 10 μM (10 μM vs 0 μM AKG, p > 0.05, Wilcoxon rank test). (B) RING assay. AKG-supplementation increased vertical climbing ability at 3 time points (20, 30, and 40 days) compared with the control diet (*p < 0.05 and **p < 0.01, two-tailed Student’s t-test). (C) Heat stress assay. AKG-treated flies were significantly more resistant to 39°C heat stress than control flies (5 μM AKG vs control, p = 0.0023, log-rank test). (D) Paraquat stress assay. There were no differences in sensitivity to paraquat stress between control and AKG-treated flies (5 μM AKG vs control, p > 0.05, log-rank test). (E) H₂O₂ stress assay. There were no differences in sensitivity to H₂O₂ stress between control and AKG-treated flies (5 μM AKG vs control, p > 0.05, log-rank test). All data are shown as the mean ± standard error of the mean (SEM).

Then, the effect of AKG on flies’ body weight was assessed over 10 days, but no significant differences were observed between AKG-treated and control flies (Supplementary Figure 2). A rapid iterative negative geotaxis (RING) assay was performed to measure flies’ climbing ability (vertical locomotion). As expected, climbing ability declined with age in both groups. However, at days 20, 30, and 40, flies fed AKG climbed significantly higher than control flies (Figure 2B), indicating that AKG improved climbing ability and enhanced physical activity.

Furthermore, we examined whether AKG-induced longevity could be in part attributable to enhanced stress tolerance. For the first 10 days of adulthood, the flies were fed diets supplemented with AKG (or control diets) and tested for tolerance to heat stress, the superoxide generator paraquat, H₂O₂, desiccation, and starvation. We found that AKG significantly increased tolerance to heat stress (Figure 2C), and this effect was accompanied by significant increases in the expression of heat shock protein genes (Hsp22 and Hsp70) compared with the control group (Supplementary Figure 3).

In contrast, AKG had no protective effect against paraquat (Figure 2D) or the oxidant H₂O₂ (Figure 2E). Accordingly, the expression of catalase (CAT) was not altered by AKG supplementation, whereas the expression of superoxide dismutase 1 (SOD1) was in fact inhibited (Supplementary Figure 4). Likewise, AKG did not affect tolerance to either desiccation or starvation (Supplementary Figure 5A, 5B). In addition, there were no significant differences in triacylglyceride (TAG) levels in flies treated with AKG compared with control flies (Supplementary Figure 5C), suggesting that lifespan extension was not associated with increased TAG levels.

AKG supplementation activates the AMPK signaling pathway in Drosophila

To examine whether the AKG-induced increase in Drosophila longevity was correlated with AMPK activation, we quantified relative steady-state levels of AMPKα and target mRNAs in flies fed AKG-supplemented or control diets. Transcript levels of AMPKα and AMPK downstream targets cry, FoxO, HNF4, p300, and Sirt1 were significantly increased, while those of HDAC4, TORC, PGC, and SREBP were significantly decreased, in AKG-fed flies compared to controls (Figure 3A). However, the expression of CREB genes (dCREB-A and dCREB-B) was comparable between groups. These results suggest that AKG supplementation induces AMPK activation and AMPK-mediated changes in gene expression in Drosophila.

Figure 3. AKG activates AMPK signaling and reduces ATP and the ATP/ADP ratio. (A) Relative mRNA levels of genes downstream of AMPK (ns = nonsignificant, p > 0.05; *p < 0.05, **p < 0.01, and ***p < 0.001; two-tailed Student’s t-test). (B) AKG-supplementation decreases ATP content in fruit flies (**p < 0.01, two-tailed Student’s t-test). (C) AKG-supplementation decreases the ATP/ADP ratio (**p < 0.01, two-tailed Student’s t-test). (D) Relative mRNA levels of genes upstream of AMPK (ns = nonsignificant, p > 0.05; *p < 0.05, **p < 0.01, and ***p < 0.001; two-tailed Student’s t-test).

Next, to verify that the increased longevity of AKG-fed flies is related to changes in cellular energy status, we measured ATP content and the ATP/ADP ratio. Consistent with increased AMPK expression, a reduction in both total ATP content and in the ATP/ADP ratio was observed in flies supplemented with AKG (Figure 3B, 3C). We next assessed the expression of three PKA subfamily genes whose products are upstream regulators of AMPK activity. Of these, Pka-C1 and Pka-R2 were significantly increased, while the expression of Pka-R1 was unaffected (Figure 3D). These findings indicate that AKG stimulated PKA expression, which might activate AMPK by enhancing the expression of LKB1 (see below). Meanwhile, the expression of CaMKII, another AMPK-activating enzyme, was also significantly increased after AKG treatment (Figure 3D).

AKG supplementation inhibits the mTOR signaling pathway

The TOR signaling pathway is a critical mediator of cell metabolism and aging. Therefore, we investigated if changes in mTOR pathway outputs were consistent with the effects of AKG on Drosophila lifespan. We found that the expression of PGC, SREBP, and the upstream genes PI3K and TORC was significantly inhibited, while that of FKBP12 and PRAS40 significantly upregulated, by AKG treatment (Figure 4A).

Figure 4. AKG supplementation increases autophagy. (A) Relative mRNA expression of genes downstream and upstream of mTOR (*p < 0.05 and **p < 0.01, two-tailed Student’s t-test). (B) Relative mRNA expression of autophagy-related genes (*p < 0.05, **p < 0.01, and ***p < 0.001, two-tailed Student’s t-test). (C) Phagosome staining. Representative fluorescence images of fly midguts stained with LysoTracker Red. Midguts of control flies (upper image) and AKG-treated flies (lower image) are shown.

It is well known that inhibition of mTOR can suppress protein synthesis and cell proliferation and increase autophagy. Consistent with the latter, we observed that AKG increased the expression of TFEB, which regulates the production of autophagosomes and lysosomes. Moreover, transcription levels of the autophagy-related genes Atg1, Atg5, Atg8a, and Atg8b were also significantly upregulated after AKG supplementation (Figure 4B). Meanwhile, phagosome staining in vitro confirmed increased phagosome formation in AKG-reared flies (Figure 4C).

AKG modifies the expression of genes associated with the Warburg effect and the JNK signaling pathway

AKG-reared flies exhibited significant upregulation of c-myc, P53, LKB1, AMPKα, and ULK1 (Figure 5A). These findings further prove that AKG increases the expression of tumor suppressor genes, inhibits the Warburg effect, and enhances physiological resistance. On the other hand, the expression of protein clearance-related genes further indicates that AKG activates AMPK and inhibits mTOR. We also assessed the fatty acid oxidation pathway by analyzing MEK and ErK expression. There was no apparent increase in the activation of MEK1/2 and ErK1/2 following AKG supplementation, suggesting that these enzymes may not be the only ones involved in the MEK and ErK pathways; perhaps other factors increase MEK1/2 and ErK1/2 kinase activity, thus increasing c-Myc expression.

Figure 5. AKG inhibits the Warburg effect and activates JNK signaling. (A) Relative mRNA expression of downstream and upstream genes involved in the Warburg effect (ns = nonsignificant, p > 0.05; *p < 0.05 and **p < 0.01; two-tailed Student’s t-test). (B) Relative mRNA expression of downstream and upstream genes in the JNK signaling pathway (*p < 0.05 and **p < 0.01, two-tailed Student’s t-test).

The C-jun N-terminal kinase (JNK) pathway, also known as the stress-activated protein kinase (SAPK) pathway, is an important branch of the MAPK pathway regulating cell cycle progression, differentiation, apoptosis, and cellular stress. We found that most genes downstream of JNK, including NFAT (NFAT4 and NFATC1) and Stat3, were markedly upregulated by AKG supplementation (Figure 5B). These findings suggest that AKG supplementation activates the JNK pathway.

Standard laboratory food

The DR medium (1x SYA) contained 100 g/L yeast (1x; MP Biomedicals, USA), 50 g/L sucrose (Tate & Lyle, UK), 15 g/L agar (Sigma-Aldrich, UK), 30 mL/L Nipagin (Chemlink Specialities, UK) and 3 mL/L propionic acid (Sigma-Aldrich, UK). This diet and its method of preparation are described in Yang et al., 2016 [32]. The fully fed medium (2x SYA) was prepared in the same way except that it contained 200 g/L yeast.

Experimental food

AKG (Sigma-Aldrich, UK) was dissolved in Milli-Q water to concentrations of 0.5, 1, 5, 10, 50, 100, 200, and 1000 μM and added to the SYA food. Milli-Q water without AKG was added to the control food.

Fly lifespan

The wild-type Dahomey (w^Dah) stock was collected in 1970 in Dahomey (now Benin). Experiments were conducted at 25°C under a 12-hour light/dark cycle at constant humidity of 65% [33, 34]. The flies were reared at a standard larval density of ~300 flies per bottle, and all enclosed adults were collected over a 12-hour period. After eclosion, the flies were allowed to mate for 48 hours, and then the females were separated under CO₂ anesthesia for further experiments. The females were then randomly allocated to the experimental food treatment groups and housed in plastic vials containing food at a density of 10 flies per vial, with 10 vials per condition (n = 100). The flies were transferred to a fresh food source 3 times per week, during which any deaths and censors were recorded. All lifespan experiments were repeated at least twice. Fly survival was evaluated with the log-rank test.

Food intake assay

One hundred female flies (10 flies per vial) per treatment (AKG or control) were reared in DR medium (1x SYA) containing 2.5% blue dye (wt/vol; Blue No. 1). After feeding for 1 hour, the flies were snap-frozen in liquid nitrogen and then homogenized in 200 mL of distilled water. An additional 800 mL of distilled water was added to the suspension, which was then filtered to remove debris and lipids through a 0.22-mm Millex filter (Millipore Corporation, Bedford, MA). The absorbance of each liquid sample was then measured at 629 nm (Thermo Scientific NanoDrop 2000c) [12, 35]. Statistical significance was determined using two-tailed Student’s t-tests.

Measurement of fecundity

Eggs were collected every 2 days for 18- to 24-hour periods during the lifespan experiments. The number of eggs laid per vial at each time point was determined by eye using a light microscope. For each condition and each time point, 10 vials were counted. Each vial contained 10 flies. Fly fecundity was evaluated with the Wilcoxon rank test.

Measurement of body weight and vertical climbing ability

Flies were reared in 10 vials (20 flies per vial; n = 200) containing an AKG-supplemented experimental diet or a control diet for 10 days. Flies were then collected under CO₂ anesthesia and weighed. Vertical climbing ability was measured by RING assay, as described in Schriner et al. 2014 [36]. At the ages of 20, 30, and 40 days, 100 flies were placed in 10 vials for each treatment group. Each vial was gently shaken until all flies had been displaced to the bottom. The flies were permitted to climb for 4 s and then photographed. Flies in both groups (AKG-fed and control) were assayed simultaneously. The assay was repeated 5 times with independent groups of flies. The distance climbed was measured in Adobe Photoshop, and the speed was calculated based on the distance/4 s. Statistical analysis was performed with two-tailed Student’s t-tests.

Stress assays

One hundred female flies (10 flies per vial) per treatment were fed for 10 days with an AKG-supplemented experimental diet or a control diet before being transferred to stress conditions. For the H₂O₂ and paraquat assays, flies were fed 5% H₂O₂ or 20 mM paraquat diluted in a 5% glucose solution supplied on filter paper, and deaths were recorded every 5 hours. For the heat stress assay, experimental flies were housed in empty vials at 39°C, and deaths were recorded every hour. For the desiccation assay, experimental flies were housed in empty vials, and deaths were recorded every 3 hours [28]. For the starvation assay, flies were housed in vials containing 1.5% agarose with no nutritional value to provide moisture, and deaths were recorded every day. Survival for all stress assays was determined by the log-rank test.

Triacylglyceride (TAG) measurement

Flies were fed an AKG-supplemented experimental diet or a control diet for 10 days before collection, and each group was divided into six replicates of 10 flies. The flies were snap-frozen in liquid nitrogen and used for TAG content measurements according to the methods of a triglyceride assay kit (Nanjing Jiancheng Bioengineering Institute). Statistical analysis was performed with two-tailed Student’s t-tests.

Measurement of ATP content and ATP/ADP ratio

Flies were fed an AKG-supplemented experimental diet or a control diet for 10 days. Each group was divided into six replicates of 10 flies. ATP content and ATP/ADP ratio were measured with an ATP content assay kit and an ATP/ADP ratio assay kit, respectively. Statistical analysis was performed with two-tailed Student’s t-tests.

LysoTracker staining, imaging, and image analysis

Flies (n = 15) were reared on an AKG-supplemented diet or a control diet for 10 days. The methods for LysoTracker staining and dissection of complete guts have been described in our previous work [12]. All images were captured with a fluorescence microscope, and we used ImageJ to analyze the number of LysoTracker-stained puncta.

Analysis of gene expression

Flies were fed for 10 days with an AKG-supplemented diet or a control diet and then frozen in liquid nitrogen for RNA quantification. Total RNA was extracted from 10 flies using TRIzol reagent, and reverse transcription was performed with a SuperScript II cDNA Synthesis Kit (Invitrogen). In addition, real-time quantitative PCR was performed using SYBR Green with RT-PCR primer pairs (Supplementary Table 1) on a Bio-Rad IQ5 (Thermo Fisher Scientific) instrument. Fold changes in expression were calculated using the 2^−ΔΔCt method and normalized to actin (housekeeping gene) levels. All measurements were performed in triplicate on each of three independent biological replicates, and data are presented as the mean ± standard deviation (SD). Statistical analysis was performed with two-tailed Student’s t-tests.

Statistical analysis

Statistical analysis was performed using GraphPad Prism software version 6.0 (GraphPad Software Inc., La Jolla, CA, USA) and SPSS software version 17.0 (SPSS Inc., Chicago, IL, USA). The tests used and the sample sizes for each experiment are indicated in the corresponding figure legends and in Materials and Methods.