Science - USA (2022-02-18)

B and C, and fig. S13, A and B). In addition,
dCTNSoverexpression was sufficient to de-
plete triglyceride stores in fed animals, where-
as they slightly accumulated after depletion of
dCTNSin the fat body upon fasting (fig. S13C).
Altogether, our data suggest that during fasting,
lysosomal cysteine mobilization is potentially

rate limiting for de novo CoA synthesis, which

in turn may promote acetyl-CoA production

throughb-oxidation of fatty acids remobilized

from triglyceride stores. The precise contribu-

tion of acetyl moieties by fatty acids and other

substrates for the synthesis of acetyl-CoA during

fasting remains to be determined.

Lysosomal-derived cysteine may control TORC1

indirectly through the flow of amino acids in

and out of the TCA cycle

Increased CoA synthesis from cysteine might

enlarge 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 through the allosteric activation of PC

by acetyl-CoA ( 25 ). To test whether increased

production of acetyl-CoA supported by lyso-

somal cysteine efflux could increase anaple-

rosis, we analyzed alanine anaplerosis in the

TCA cycle in animals overexpressingdCTNS

in the fat body. We supplemented a low-protein

diet with a [U-^13 C]alanine tracer and followed

theanapleroticfluxofalanineintheTCAcycle

in dissected fat bodies (Fig. 5D). We used a

tracer amount that had a negligible contri-

bution to the total alanine pool and also did

not affect cysteine metabolism to acetyl-CoA

(fig.S14,AandB).dCTNSoverexpression

increased (by more than twofold) alanine

anaplerosis as well as oxidative flux in the

TCA cycle through citrate synthase (that con-

sumes OAA and acetyl-CoA to generate citrate;

Fig. 5E). We therefore propose that during

fasting, 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

and isocitrate.

During fasting, given the fixed and prees-

tablished level of carbons available, increased

abundance of TCA cycle intermediates may

indicate the retention of anaplerotic inputs in

the TCA cycle at the expense of their extrac-

tion 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 re-

cycling on individual amino acid pools.dCTNS

overexpression in the fat body led to depletion

of aspartate and downstream nucleotide pre-

cursors (IMP and uridine 5′-monophosphate),

as well as, to a lesser extent, asparagine and

glutamate (Fig. 6A and fig. S15, A and B). The

abundances of aspartate and IMP were in-

creased in fasteddCTNS−/−animals, and cys-

teamine treatment partially normalized their

concentration (fig. S15C). Aspartate is a cata-

plerotic product of OAA ( 1 ), a process that in-

volves glutamate oxaloacetate transaminase 2

(Got2). Cysteine metabolism may transient-

ly trap anaplerotic carbons into the TCA cycle

in fasted animals, away from their imme-

diate extraction for biosynthesis. We analyzed

whether cysteine metabolism could regulate

growth and antagonize TORC1 reactivation

through limiting the availability of glutamate

and aspartate, because these amino acids are

critical regulators of cell growth and have

Jouandinet al.,Science 375 , eabc4203 (2022) 18 February 2022 6 of 11

C

contro

l

dCTNS

-/-

cont

rol

dCT

NS

• /-

Carnitine Myristoyl-carnitine Dodecanoyl-carnitine Octanoyl-carnitine Butyryl-carnitine

B

control

Acetyl-CoA

(whole larvae)

(Fold change to control)

ns *

lpp>

Fed Fast (8h)

Cysteine

Fatty Acids

CoA

Acetyl-CoA

Beta

Oxidation

Fasting fat body

dCTNS

A

lpp>

Fed Fast Fed Fast Fed Fast Fed Fast Fed Fast

Acetyl-CoA

(whole larvae)

Fed Fast (8h)

contro

l

dCTNS

-/-

controldCTNS

• /-

ns ****

0

0.5

1.0

1.5

0

0.5

1.0

1.5

0

0.5

1.0

1.5

0

0.5

1.0

1.5

0

0.5

1.0

1.5

0

0.5

1.0

1.5

2.0

0

0.5

1.0

1.5

2.0

2.5

0

0.5

1.0

1.5

2.0

2.5

0

1

2

3

0

0.5

1.0

1.5

2.0

2.5

control

dCTNS

-/-

contro

l

dCT

NS

-/-

control

dCTNS

-/-

control

dCT

NS

-/-

control

dCTNS

-/-

control

dCTNS

-/-

control

dCTNS

-/-

control

dCTNS

-/-

(Fold change to control)

Fed Fast Fed Fast Fed Fast Fed Fast Fed Fast

controldCTNScontro

l

dCT

NS

contro

l

dCTNScontroldCT

NS

controldCTNScontro

l

controldCTNScon dCTNS

trol

controldCTNScontroldCTNS dCTNS

*

***

ns

*** *** ***

***

**

* ****

*** *** **

**

ns

ns

ns

ns ns

ns

**

DE

Pyruvate

Citrate

aKG

Malate

OAA

Acetyl-CoA

Cysteine

PC

Alanine

Fumarate

Succinate

dCTNS

Alanine m+3Lac

tate m+3

OAAMala

te

Succinate

CitrateaKG

Suc

cinate

Fumar

ate

Fold change to control

Alanine f lux ratio lpp>dCTNS / control

in dissected fat body

Acetyl-CoA

(Fold change to control)

Acetyl-CoA

(Fold change to control)

Carnitines

0

0.5

1.0

1.5

2.0

2.5

0

1

2

3

Carnitines

contr

ol

dCTNS dCTNS

ns ns

ns

********

****

***

**

**

0

1

2

3

4

5

6

Fig. 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.
(BandC) Metabolite levels in whole third-instar larvae showingdCTNS−/−anddCTNSoverexpression
in the fat body. (D) Schematic of alanine carbon flux into the TCA cycle upon fasting. (E) Alanine
flux ratio. Shown is the fold changelpp>dCTNS/control (lpp>attp40) for the indicated TCA cycle
intermediates isotopomers measure by LC-MS/MS in dissected fat bodies from third-instar larvae
fed a low-protein diet with 25 mM U-^13 C-alanine for 6 hours.

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