Lysosomal cystine mobilization shapes the response of TORC1 and tissue growth to fasting

Abstract

Adaptation to nutrient scarcity involves an orchestrated response of metabolic and signaling pathways to maintain homeostasis. We find that in the fat body of fasting Drosophila, lysosomal export of cystine coordinates remobilization of internal nutrient stores with reactivation of the growth regulator target of rapamycin complex 1 (TORC1). Mechanistically, cystine was reduced to cysteine and metabolized to acetyl-coenzyme A (acetyl-CoA) by promoting CoA metabolism. In turn, acetyl-CoA retained carbons from alternative amino acids in the form of tricarboxylic acid cycle intermediates and restricted the availability of building blocks required for growth. This process limited TORC1 reactivation to maintain autophagy and allowed animals to cope with starvation periods. We propose that cysteine metabolism mediates a communication between lysosomes and mitochondria, highlighting how changes in diet divert the fate of an amino acid into a growth suppressive program.

Figure 1:. TORC1 reactivation upon prolonged fasting correlates with increase of TCA cycle intermediates.

(A) Prolonged fasting leads to TORC1 reactivation. Phosphorylation levels of the direct TORC1 target S6K in dissected fat bodies from fasting larvae (i.e. placed on a tissue soaked in PBS). (B) Heat map metabolite levels (LC-MS/MS) from whole mid-third-instar larvae (left) or dissected fat bodies (right), fed (0h) and fasted. Lower panels are individual plots from the same dataset. (C) Schematic of anaplerosis through PC. (D) Knockdown of pcb/PC suppresses TORC1 reactivation upon prolonged fasting. For (B) and (D), data are shown as mean +/− SD. ns, P≥ 0.05; *, P≤ 0.05; **, P≤ 0.01; ***, P≤ 0.005; ****P ≤ 0.0001 (see the material and methods for details).

Figure 2:. Lysosome-derived cysteine promotes elevation of TCA cycle intermediates and antagonizes TORC1 reactivation.

(A) Amino acid screen reveals cysteine as a growth suppressor. Time to pupariation for larvae fed a low-protein diet supplemented with the indicated amino acids all along development (see supplementary materials and methods for concentrations; the cysteine concentration is 5 mM). Scale bar, 1 mm. (B) Cysteine levels in whole control larvae (lpp>attp40). N = 5. (C) Schematic of lysosomal cystine (CySS) efflux through cystinosin/ CTNS. (D) Loss of Drosophila cystinosin in the fat body (lpp>dCTNS RNAi, dCTNS-i) leads to higher TORC1 reactivation upon prolonged fasting. P-S6K levels in dissected fat bodies from larvae fasted for the indicated time. lpp>w RNAi, control background. (E) dCTNS maintains autophagy during fasting. dCTNS^−/− clones (non-GFP, outlined) in 80h AEL larvae expressing mCherry-Atg8a fasted for 8h. Scale bar, 10 μm. (F) dCTNS overexpression suppresses TORC1 activity. P-S6K levels in dissected fat bodies from larvae fasted for the indicated time. lpp>GFP RNAi (GFP-i), control background. (G) dCTNS overexpression induces autophagy. dCTNS overexpression clones (GFP marked, outlined) in 80h AEL larvae. Scale bar 10 μm. (H) dCTNS regulates TCA cycle intermediates levels upon fasting. Relative metabolites levels measured by LC-MS/MS in 80h AEL larvae fed or fasted for 8h. Controls are dCTNS^+/− (upper graphs) or GFP-i (lower graphs). For (B), (D), (F) and (H), data are shown as mean +/− SD. ns, P≥ 0.05; **, P≤ 0.01; ***, P≤ 0.005; ****P≤ 0.0001 (see the materials and methods for details).

Figure 3:. dCTNS controls resistance to starvation through cysteine efflux and TORC1.

(A) dCTNS does not affect life span in fed condition. Life span of control (w¹¹¹⁸) and dCTNS^−/− animals fed a standard diet. N = 2. (B) dCTNS in the fat body controls starvation resistance during development. Shown is the fold change time to pupariation for larvae of indicated genotype grown on control (fed) or low-protein diet. Controls are dCTNS+/− (left panel) or white RNAi (control-i, right panel). (C) dCTNS controls starvation resistance of adult animals. Survival of control (w¹¹¹⁸) and dCTNS^−/− animals fed a chemically defined starved diet composed only of physiologically relevant ions, including bio-metals (see the materials and methods). (D to F) Low dose of rapamycin and cysteine treatments rescue starvation sensitivity of dCTNS^−/− animals. Show is the developmental time of larvae raised on a low-protein diet supplemented with the indicated concentration of rapamycin (D) or 0.1 mM cysteine (F) and survival of adult flies on chemically defined starved diet with or without 1 mM cysteine (E). Controls are dCTNS^+/− [(D) and (F)] and w¹¹¹⁸ (E). (G) Cysteamine treatment restores starvation resistance of dCTNS^−/− animals. Show is the life span of control (w¹¹¹⁸) and dCTNS^−/− animals fed a chemically defined starved diet supplemented with 0.5 mM cysteamine or vehicle. For (B) to (G), data are shown as mean +/− SEM. ns, P≥ 0.05; *, P≤ 0.05; **, P≤ 0.01; ***, P≤ 0.005; ****, P≤ 0.0001 (see the materials and methods for statistics details).

Figure 4:. Cysteine fuels de novo CoA/acetyl-CoA metabolism.

(A) Mean fractional enrichment +/− SD of U-¹³C-cysteine (N = 10), ¹³C₃_¹⁵N₁-cysteine (N = 5) or unlabeled samples (N = 10) in indicated metabolites measured by LC-MS/MS in whole larvae fast overnight with 5 mM tracer. m+n refers to the number of ¹³C atoms (+n) added to the expected mass spectra of each measured isotopomer (m); m+0 means unlabeled. (B) Schematic of cysteine metabolism and labelling patterns from U-¹³C-cysteine and ¹³C₃-¹⁵N₁-cysteine tracers. Red arrows indicate main cysteine flux.

Figure 5:. Cysteine metabolism to acetyl-CoA affects the concentration of fatty acids and increases carbon flux through PC and the TCA cycle.

(A) Schematic of acetyl-CoA synthesis during fasting. (B and C) Metabolite levels in whole 3^rd instar larvae showing dCTNS^−/− and dCTNS overexpression in the fat body. (D) Schematic of alanine carbon flux into the TCA cycle upon fasting. E) Alanine flux ratio. Shown in the fold change lpp>dCTNS /control (lpp>attp40) for the indicated TCA cycle intermediates isotopomers measure by LC-MS/MS in dissected fat bodies from 3^rd instar larvae fed a low-protein diet with 25 mM U-¹³C-alanine for 6 hours.

Figure 6:. Cysteine metabolism regulates TORC1 and growth through cataplerotic amino acids levels.

(A) Relative levels of aspartate in 85h AEL larvae dCTNS^−/− (whole larvae and fat body) and after dCTNS overexpression in the fat body. (B) Amino acid supplementation suppresses the developmental delay induced by dCTNS overexpression in the larval fat body. Shown is the fold change time to pupariation for larvae fed a low-protein diet with or without supplementation with the indicated amino acids (Ala, Pro and Glu: 5 mM; Asp: 10 mM). Red asterisks show the significance between dCTNS-overexpressing animals treated with vehicle vs. amino acids. (C) Photographs of aged matched animals fed a low-protein diet with or without 5 mM cysteine with or without the indicated metabolites (25 mM each). Scale bar, 1 mm. (D) dCTNS-induced TORC1 inhibition is reversed by supplementation with the indicated amino acids. P-S6K levels in fat body from fed and fasted (6h) larvae of indicated genotypes. The 70 to 72h AEL larvae were transferred to a low-protein diet with or without supplementation with the indicated amino acids (Ala and Pro: 20 mM; Asp and Glu: 10 mM). Control is GFP-i. For (A), (B), and (D), data are shown as mean +/− SD. ^ns, P≥ 0.05; *, P≤ 0.05; **, P≤ 0.01; ***, P≤ 0.005; ****P≤ 0.0001 (see the materials and methods for details).