Abstract
Adaptation to nutrient scarcity involves an orchestrated response of metabolic and signaling pathways to maintain homeostasis. We provide evidence that lysosomal export of cystine coordinates remobilization of internal nutrient stores with reactivation of the growth regulator TORC1 signaling upon fasting in the Drosophila fat body. Mechanistically, cystine is reduced to cysteine and metabolized to acetyl-CoA by consuming lipids. In turn, acetyl-CoA retains carbons from alternative amino acids in the form of TCA cycle intermediates, thereby restricting amino acids availability, notably aspartate. This process limits TORC1 reactivation to maintain autophagy and allows animals to cope with starvation periods. We propose that cysteine metabolism mediates a communication between lysosomes and mitochondria to maintain the balance between nutrient supply and consumption under starvation, highlighting how changes in diet divert the fate of an amino acid into a growth suppressive program.
One Sentence Summary Lysosomal cysteine recycling is a metabolic break that maintains autophagy upon starvation through coenzyme A synthesis.
Main Text
Organisms constantly cope with variations in diet by adjusting metabolism, and specific organs impact systemic nutrient homeostasis. Upon fasting, the liver remobilizes nutrients through gluconeogenesis and β-oxidation of fatty acids to support peripheral tissue function (1, 2). Variation in nutrient availability induces parallel changes in signaling pathways activity, which resets intracellular metabolic turnover. The TORC1 (Target of Rapamycin Complex 1) signaling pathway integrates sensing of amino acids and other nutrients with signals from hormones and growth factors to promote growth and anabolism (3). Nutrient scarcity inhibits TORC1 to limit growth and promote catabolic programs, including autophagy that recycles internal nutrient stores to promote survival (4). Autophagy involves the sequestration of cytosolic material into autophagosomes that fuse with lysosomes for cargo degradation and recycling. The lysosomal surface is also the site where nutrient and growth factors sensing pathways converge to activate TORC1. Degradation within lysosomes generates new amino acids that in turn fuel metabolic pathways, including the tricarboxylic acid (TCA) cycle and gluconeogenesis, and reactivate TORC1, altogether maintaining minimal anabolism and growth (5-8). However, how organisms regulate the limited pools of remobilized nutrients and balance homeostatic metabolism with anabolic TORC1 activity over the course of starvation is poorly understood.
Results
To study the regulation of metabolism and TORC1 signaling in vivo, we used the Drosophila larval fat body, an organ analogous to the liver and adipose tissue in mammals. The fat body responds to variation in nutrient availability through TORC1 signaling, which in turn regulates systemic larval growth rate through the secretion of growth factors from distant organs (9, 10). When larvae were completely deprived of their food source (but kept otherwise hydrated), TORC1 signaling in the fat body was acutely downregulated at the onset of starvation, as expected. However, prolonged starvation led to partial and progressive reactivation of TORC1 over time (Fig. 1A). This process was dependent on autophagy induction (Fig. S1A, B), consistent with autophagy regulating amino acid recycling and TORC1 reactivation in mammalian cells (6, 8). To understand how changes in nutrients and metabolites intersect with mTORC1 signaling during this fasting response, we initially took two complementary unbiased approaches: 1) a targeted mass-spectrometry based screen for polar metabolites altered during starvation and 2) a larval growth screen to test the effects of individual amino acids during starvation.
Metabolic profiling of whole animals in matching conditions revealed depletion of most metabolites over the course of starvation (Fig. 1B). By contrast, TCA cycle intermediates accumulated over time, in particular citrate/ isocitrate. This was not due to defective TCA cycle activity upon fasting evidenced by functional U-13C6-glucose oxidation in the TCA cycle and NADH/NAD+ ratios at equilibrium between fed and fast conditions (Fig. S1C, D). Similar metabolomics profiles were observed in fat bodies dissected out from fed and starved animals (Fig. 1B), indicating that TORC1 reactivation in the fat body correlates with accumulation of TCA cycle intermediates, in particular citrate / isocitrate. To test a link between changes in the TCA cycle and TORC1 reactivation, we targeted pyruvate carboxylase (PC). PC serves an anaplerotic function by replenishing TCA cycle intermediates through oxaloacetate (Fig. 1C), a process that regulates gluconeogenesis and regeneration of amino acids (1). Knockdown of PC (pcb/CG1516) in the larval fat body elevated alanine levels (Fig. S1E), consistent with alanine as an important PC substrate (1). However, it suppressed TORC1 reactivation upon fasting (Fig. 1D), suggesting a functional link between the TCA cycle and TORC1 signaling upon fasting.
In our second screen, we tested the effects of single amino acid supplementation on larval growth upon fasting (see Supplementary Materials). Unexpectedly, this screen revealed cysteine as a unique amino acid associated with growth suppressive properties (Text S1; Fig. 2A; Fig. S2A-D). Again, this process involved changes in TORC1 signaling as cysteine treatment partially inhibited TORC1 activity in fat bodies and constitutive activation of TORC1, in the absence of the TORC1 regulator Gator1 (nprl2-/-) (11), partially restored growth under cysteine supplementation (Fig. S2E, F).
The results of our two screens led us to explore the relationship between cysteine metabolism, the TCA cycle, and TORC1 during the fasting response. Importantly, we found that cysteine levels increased upon fasting (Fig. 2B), consistent with previous reports in yeast (12), and that cysteine treatment increased the levels of TCA cycle intermediates, particularly upon fasting (Fig. S2G). To further understand this effect of cysteine, we searched for its intracellular source under starvation conditions. Physiologically, cellular cysteine can either be synthesized in the cytosol or transported as cystine from the extracellular space by the Xc- antiporter (13, 14) and by cystinosin from the lysosome (15) (Fig. 2C). Abolishing autophagy in the fat body decreased cysteine levels upon fasting (Fig. S3A), suggesting that lysosomal function regulates cysteine balance. Thus, we analyzed the role of the lysosomal cystine transporter cystinosin in recycling cysteine upon fasting. Cystinosin, encoded by CTNS in mammals, is mutated in the lysosomal storage disorder cystinosis and has previously been implicated in mTORC1 signaling and autophagy (15-20). Endogenous tagging of the Drosophila ortholog CG17119 (hereafter referred to as dCTNS) confirmed its specific lysosomal localization in fat body cells. dCTNS-/- larvae showed accumulation of cystine (Fig. S3B-D), consistent with a role for cystinosin in lysosomal cystine transport. In fed conditions, control and dCTNS-/- larvae showed similar cysteine levels, likely reflecting dietary intake as the main source of cysteine. By contrast, upon fasting, cysteine levels dropped in dCTNS -/- larvae (Fig. S3E). Conversely, dCTNS overexpression increased cysteine levels, which caused a developmental delay similar to cysteine treatment (Fig. S3F, G). We therefore concluded that cystinosin recycles cysteine from the lysosome upon fasting and analyzed its effect on TORC1 reactivation and the TCA cycle. While dCTNS knockdown in larval fat body did not affect TORC1 inhibition at the onset of starvation, it slightly increased TORC1 reactivation upon prolonged starvation (Fig. 2D). Analysis of dCTNS-/- fat body clones showed that increased TORC1 signaling was cell-autonomous and sufficient to compromise maintenance of autophagy in starvation. Accordingly, treatment with the TORC1 inhibitor rapamycin was able to restore autophagy in dCTNS-deficient cells (Fig. 2E, Fig. S3H, I). Conversely, dCTNS overexpression caused downregulation of TORC1 and induced autophagy (Fig. 2F, G). Metabolic profiling of dCTNS-/- animals showed a depletion of TCA cycle intermediates specifically upon fasting. In contrast, they accumulated following overexpression of dCTNS in fed and starved animals (Fig. 2H).
To test the physiological importance of cysteine recycling upon fasting, we analyzed the role of dCTNS on starvation resistance. In fed condition, dCTNS deficiency in fat body cells neither affected larval development nor the lifespan of adult flies (Fig. 3A, B). However, in fasted animals, dCTNS deficiency caused a developmental delay and shortened lifespan (Fig. 3B, C). This starvation sensitivity was rescued by low concentrations of rapamycin, indicating a key role for altered TORC1 signaling and autophagy in mediating dCTNS growth phenotypes (Fig. 3D and see text S2). The role of dCTNS upon fasting was dependent on its cystine transport function as treatment with either cysteamine (which exports cystine out of the lysosome independently of cystinosin (21)) or low concentrations of cysteine (that did not affect development of control animals) rescued the starvation sensitivity of dCTNS-/- animals (Fig. 3E-G). Altogether, we demonstrate the physiological importance of cysteine recycling from the lysosome upon fasting, which supports the elevation of TCA cycle intermediates and antagonizes TORC1 reactivation to maintain autophagy.
Next, we sought a mechanistic link between cysteine metabolism and the TCA cycle and performed cysteine tracing experiments. For this, we supplemented a fasted diet with either U-13C-cysteine or 13C3 15N1-cysteine. Consistent with cysteine metabolism in mammals, this revealed three major cysteine fates: glutathione (GSH), taurine, and coenzyme A (CoA) (Fig. 4A; S4A). The CoA derived from labeled cysteine also contributed significantly to the formation of acetyl-CoA in the fat body (Fig. 4A, B; S4B). Under fasting conditions, the acetyl group of this acetyl-CoA pool is mainly provided through β-oxidation of fatty acids (Fig. 5A). Accordingly, dCTNS overexpression upon fasting elevated the level of acetyl-CoA and decreased the level of acyl-carnitines and fatty acids. This phenotype was mirrored in fasted dCTNS KO animals (Fig. 5B, C; S5A), suggesting that during fasting, lysosomal cysteine mobilization is rate limiting for de novo CoA metabolism and supports acetyl-CoA synthesis through β-oxidation.
Increased CoA synthesis from cysteine could play a central role in enlarging the TCA cycle carbon pool in at least two ways. First, by providing the CoA required to accept increasing amounts of carbon from fatty acids to form acetyl-CoA, and second, by promoting anaplerosis of alternative carbon sources via the acetyl-CoA-dependent allosteric activation of PC (22). To test whether the increased production of acetyl-CoA supported by lysosomal cysteine efflux could elevate anaplerosis and TCA cycle intermediates, we analyzed alanine anaplerosis in the TCA cycle following dCTNS overexpression. We supplemented a fasted diet with [U-13C]alanine so that the tracer had negligible contribution to the total alanine pool (Fig. S5B), and followed the anaplerotic flux of alanine in the TCA cycle in dissected fat body (Fig. 5D). We found that dCTNS overexpression strongly increased (>2 fold) alanine anaplerosis as well as oxidative flux in the TCA cycle, through citrate synthase (that consumes OAA and acetyl-CoA to generate citrate, Fig. 5E). Altogether, we propose that during starvation, cysteine recycling and metabolism to acetyl-CoA in the fat body supports anaplerosis through PC and flux through citrate synthase, thereby contributing to the accumulation of TCA cycle intermediates, in particular citrate/ isocitrate.
Finally, we addressed a link between the level of TCA cycle intermediates and TORC1 reactivation. Upon starvation, given the fixed and pre-established level of carbons available, elevation of the level of TCA cycle intermediates may indicate the retention of anaplerotic inputs in the TCA cycle at the expense of their extraction for biosynthesis (i.e. cataplerosis). Because cataplerosis promotes amino acid synthesis (1), which in turn affects TORC1 signaling (6), we analyzed the effect of lysosomal cysteine recycling on individual amino acid pools. Interestingly, dCTNS overexpression in the fat body led to depletion of aspartate and downstream nucleotide precursors (IMP, UMP), as well as, to a lesser extent, asparagine and glutamate (Fig. 6A, S6A, B). Conversely, aspartate and UMP were elevated in dCTNS-/- animals upon fasting. Aspartate is a cataplerotic product of alanine/oxaloacetate (1), a process that involves Got2. Thus, data suggest that cysteine metabolism transiently traps anaplerotic carbons into the TCA cycle upon fasting, away from their immediate extraction for biosynthesis. Further studies will be required to understand the mechanistic basis of this process, which could involve an effect of acetyl-CoA on oxaloacetate flow through citrate synthase towards the TCA cycle vs. Got2 and aspartate synthesis upon fasting. The mild effect on glutamate remains unclear at this point, and may be connected to the metabolism of aspartate as glutamate serves as nitrogen donor for transamination through Got2 during aspartate synthesis. Nevertheless, we analyzed whether cysteine metabolism could regulate growth and antagonize TORC1 reactivation at least through limiting the availability of glutamate and aspartate, because these amino acids are critical regulators of cell growth and have previously been implicated in TORC1 signaling (6, 23). To do so, we aimed at replenishing aspartate and glutamate levels in dCTNS overexpressing animals using dietary supplementation with anaplerotic amino acids in excess. We found that combinatorial treatments with alanine, aspartate, glutamate and proline (that replenishes glutamate in flies(24)) restored normal levels of aspartate and glutamate without compromising dCTNS-induced elevation of TCA intermediates (Fig. S6C, D). Strikingly, these treatments rescued the developmental delay induced by dCTNS overexpression in the fat body (Fig. 6B). Similarly, co-supplementation of cysteine with excess of single amino acids including alanine, aspartate, glutamate and proline each rescued cysteine-induced growth suppression upon fasting (Fig. 6C). Finally, combinatorial amino acid treatments restored TORC1 activity upon fasting following dCTNS overexpression in the fat body (Fig. 6D). Consistent with the importance of aspartate synthesis, loss of Got2 blocked cell growth, induced autophagy and affected TORC1 targets (Fig. S6E), in line with previous reports (6). Altogether, we show that cysteine metabolism regulates anaplerotic carbon flow in the TCA cycle and the level of amino acids, in particular aspartate. We propose that cysteine converts the TCA cycle into a reservoir of carbons while limiting their extraction for biosynthesis. This process spares nutrients to survive a starvation period while resetting TORC1 reactivation to a threshold that maintains minimal growth without compromising autophagy.
Discussion
Maintaining cellular homeostasis upon nutrient shortage is an important challenge for all animals. On the one hand, downregulation of mTORC1 is necessary to limit translation, reduce growth rates and engage autophagy. On the other hand, minimal mTORC1 activity is required to promote lysosomes biogenesis, thus maintaining autophagic degradation necessary for survival (8). Here, we show that TORC1 reactivation upon fasting integrates biosynthesis of amino acids from anaplerotic inputs into the control of growth. The regulation of at least aspartate levels appears critical during this process, likely because it serves as precursor for various macromolecules including other amino acids and nucleotides, which in turn impinge on TORC1 activity (25). We identify another critical layer of regulation, whereby cysteine recycling via the lysosome fuels acetyl-CoA synthesis and prevents reactivation of TORC1 above a threshold that would compromise autophagy and survival upon fasting. We show that reactivation of TORC1 upon fasting is not passively controlled by the extent of amino acid remobilized from the lysosome. Instead, cysteine metabolism supports an increased incorporation of the carbons from these remobilized amino acids into the TCA cycle. We propose that the remobilized amino acids are transiently stored in the form of TCA cycle intermediates compartmentalized in the mitochondria, thereby regulating their accessibility to maintain autophagy over a starvation period. Interestingly, this pathway is self-regulated by autophagy, as autophagic protein degradation controls cystine availability through the lysosomal CTNS transporter. Thus, in contrast to fed conditions where amino acid transporters at the plasma membrane maintain high cytosolic levels of leucine and arginine that can directly be sensed by members of the TORC1 machinery (3), TORC1 reactivation in prolonged starvation is regulated indirectly by lysosome-mitochondrial crosstalk.
Multiple functions of cysteine impinge on cellular metabolism, including tRNA thiolation, the generation of hydrogen sulfide, the regulation of HIF transcription factors, and its antioxidant function through glutathione synthesis (26-28). Here, we show that CoA is a main fate of cysteine that affects oxidative metabolism in the mitochondria, which is source of reactive oxygen species (ROS). Thus, we speculate that the antioxidant function of cysteine could be coupled to its effects on the mitochondria to buffer ROS production. Supplementation with cysteine or modified molecules such as N-acetyl-cysteine (NAC) can be used to efficiently buffer oxidative stress, a process under current investigation to alleviate symptoms of a variety of diseases involving oxidative stress or glutathione deficiency, including cystinosis (29-32). In addition, cysteine or NAC extends lifespan in flies, worms and mice, and mice fed NAC show a sudden drop in body weight, similar to dietary restriction (33, 34). Our results may challenge the idea that cysteine acts strictly through its antioxidant function since we demonstrate that cysteine supplementation restricts the availability of particular amino acids and limits mTOR activity - processes known to extend lifespan.
In summary, we demonstrate that cysteine metabolism acts in a feedback loop involving de novo coenzyme A synthesis, the TCA cycle and amino acid metabolism to limit mTORC1 reactivation upon prolonged fasting and maintain autophagy. We show that this pathway is needed for developing organisms to balance growth and survival during periods of food shortage.
Funding
The Cystinosis Research Foundation (to P.J., Z.M., M.S. and N.P.), the LAM Foundation Fellowship Award LAM00105E01-15 (to A.P.), National Institutes of Health 5P01CA120964-04 (to J.M.A. and N.P.) and R01AR057352 (to N.P), the ATIP-Avenir program, the Fondation Bettencourt-Schueller (Liliane Bettencourt Chair of Developmental Biology) as well as State funding by the Agence Nationale de la Recherche (ANR) under the “Investissements d’avenir” program (ANR-10-IAHU-01) and the NEPHROFLY (ANR-14-ACHN-0013) grant (to MS.). N.P. is an investigator of the Howard Hughes Medical Institute. National Institutes of Health and National Cancer Institute (R00-CA194314) (to C.C.D); V Foundation For Cancer Research, V Scholar Grant 2019 (V2019-009) (to C.C.D).
Author contributions
P.J., Z.M., C.D., M.S. and N.P designed the experiments; P.J., Z.M., A.P., M.D., J.A., and I.N performed the experiments; P.J., Z.M., M.S. and N.P. participated in interpretation of data and P.J wrote the manuscript with inputs from Z.M., C.C.D, M.S. and N.P.
Competing interests
Authors declare no competing interests.
Data and materials availability
All data is available in the main text or the supplementary materials.
Supplementary Materials
Materials and Methods
Supplementary Text S1, S2
Figures S1-S6
Tables S1
Acknowledgements
We thank Eric Baehrecke, Christen Mirth, Pierre Léopold, Aurelio Teleman, Frederik Wirtz-Peitz, Yohanns Bellaïche, Isabelle Gaugue, Sylvia Sanquer, Anne-Claire Boschat, Mary A. Lilly, the TRIP (http://www.flyrnai.org/TRiP-HOME.html), BDSC and VDRC stock centers for providing stocks and reagents. We thank Corinne Antignac, Lewis C. Cantley, Bruno Gasnier, Joshua D. Rabinowitz and David M. Sabatini for comments on the manuscript. We thank the Imagine Microscopy platform for assistance with microscopy.
Footnotes
This revised version clarifies the mechanistic basis of cysteine metabolism to acetyl-CoA.