l-Asparaginase regulates mTORC1 activity via a TSC2-dependent pathway in pancreatic beta cells

1. N. Hashimoto, Y. Kido, T. Uchida, S. Asahara, Y. Shigeyama, T. Matsuda, A. Takeda, D.

Tsuchihashi, A. Nishizawa, W. Ogawa, Y. Fujimoto, H. Okamura, K.C. Arden, P.L. Herrera,

T. Noda, M. Kasuga, Ablation of PDK1 in pancreatic beta cells induces diabetes as a result of

loss of beta cell mass, Nat. Genet. 38 (2006) 589–593. https://doi.org/10.1038/ng1774.

2. T. Matsuda, Y. Kido, S. Asahara, T. Kaisho, T. Tanaka, N. Hashimoto, Y. Shigeyama, A.

Takeda, T. Inoue, Y. Shibutani, M. Koyanagi, T. Hosooka, M. Matsumoto, H. Inoue, T.

Uchida, M. Koike, Y. Uchiyama, S. Akira, M. Kasuga, Ablation of C/EBPbeta alleviates ER

stress and pancreatic beta cell failure through the GRP78 chaperone in mice, J. Clin. Invest.

120 (2010) 115–126. https://doi.org/10.1172/jci39721.

3. S. Asahara, Y. Shibutani, K. Teruyama, H.Y. Inoue, Y. Kawada, H. Etoh, T. Matsuda, M.

Kimura-Koyanagi, N. Hashimoto, M. Sakahara, W. Fujimoto, H. Takahashi, S. Ueda, T.

Hosooka, T. Satoh, H. Inoue, M. Matsumoto, A. Aiba, M. Kasuga, Y. Kido, Ras-related C3

botulinum toxin substrate 1 (RAC1) regulates glucose-stimulated insulin secretion via

modulation of F-actin, Diabetologia 56 (2013) 1088–1097. https://doi.org/10.1007/s00125013-2849-5.

4. S. Asahara, H. Etoh, H. Inoue, K. Teruyama, Y. Shibutani, Y. Ihara, Y. Kawada, A.

Bartolome, N. Hashimoto, T. Matsuda, M. Koyanagi-Kimura, A. Kanno, Y. Hirota, T.

Hosooka, K. Nagashima, W. Nishimura, H. Inoue, M. Matsumoto, M.J. Higgins, K. Yasuda,

N. Inagaki, S. Seino, M. Kasuga, Y. Kido, Paternal allelic mutation at the Kcnq1 locus

reduces pancreatic β-cell mass by epigenetic modification of Cdkn1c, Proc. Natl. Acad. Sci.

U. S. A. 112 (2015) 8332–8337. https://doi.org/10.1073/pnas.1422104112.

5. K. Miyake, W. Yang, K. Hara, K. Yasuda, Y. Horikawa, H. Osawa, H. Furuta, M.C.Y. Ng,

Y. Hirota, H. Mori, K. Ido, K. Yamagata, Y. Hinokio, Y. Oka, N. Iwasaki, Y. Iwamoto, Y.

Yamada, Y. Seino, H. Maegawa, A. Kashiwagi, H.Y. Wang, T. Tanahashi, N. Nakamura, J.

Takeda, E. Maeda, K. Yamamoto, K. Tokunaga, R.C.W. Ma, W.Y. So, J.C.N. Chan, N.

Kamatani, H. Makino, K. Nanjo, T. Kadowaki, M. Kasuga, Construction of a prediction

model for type 2 diabetes mellitus in the Japanese population based on 11 genes with strong

evidence of the association, J. Hum. Genet. 54 (2009) 236–241.

https://doi.org/10.1038/jhg.2009.17.

6. T.E. Dever, A.G. Hinnebusch, GCN2 whets the appetite for amino acids. Mol. Cell 18

(2005) 141–142. https://doi.org/10.1016/j.molcel.2005.03.023.

7. A. Kanno, S.I. Asahara, A. Furubayashi, K. Masuda, R. Yoshitomi, E. Suzuki, T. Takai, M.

Kimura-Koyanagi, T. Matsuda, A. Bartolome, Y. Hirota, N. Yokoi, Y. Inaba, H. Inoue, M.

Matsumoto, K. Inoue, T. Abe, F.Y. Wei, K. Tomizawa, W. Ogawa, S. Seino, M. Kasuga, Y.

Kido, GCN2 regulates pancreatic β cell mass by sensing intracellular amino acid levels, JCI

Insight 5 (2020) e128820. https://doi.org/10.1172/jci.insight.128820.

8. A. Kanno, S.I. Asahara, K. Masuda, T. Matsuda, M. Kimura-Koyanagi, S. Seino, W.

Ogawa, Y. Kido, Compensatory hyperinsulinemia in high-fat diet-induced obese mice is

associated with enhanced insulin translation in islets, Biochem. Biophys. Res. Commun. 458

(2015) 681–686. https://doi.org/10.1016/j.bbrc.2015.02.024.

9. Y. Shigeyama, T. Kobayashi, Y. Kido, N. Hashimoto, S. Asahara, T. Matsuda, A. Takeda, T.

Inoue, Y. Shibutani, M. Koyanagi, T. Uchida, M. Inoue, O. Hino, M. Kasuga, T. Noda,

Biphasic response of pancreatic beta-cell mass to ablation of tuberous sclerosis complex 2 in

mice, Mol. Cell. Biol. 28 (2008) 2971–2979. https://doi.org/10.1128/mcb.01695-07.

10. M. Koyanagi, S. Asahara, T. Matsuda, N. Hashimoto, Y. Shigeyama, Y. Shibutani, A.

Kanno, M. Fuchita, T. Mikami, T. Hosooka, H. Inoue, M. Matsumoto, M. Koike, Y.

Uchiyama, T. Noda, S. Seino, M. Kasuga, Y. Kido, Ablation of TSC2 enhances insulin

secretion by increasing the number of mitochondria through activation of mTORC1, PLoS

One 6 (2011) e23238. https://doi.org/10.1371%2Fjournal.pone.0023238.

11. A. Bartolomé, M. Kimura-Koyanagi, S. Asahara, C. Guillén, H. Inoue, K. Teruyama, S.

Shimizu, A. Kanno, A. García-Aguilar, M. Koike, Y. Uchiyama, M. Benito, T. Noda, Y. Kido,

Pancreatic β-cell failure mediated by mTORC1 hyperactivity and autophagic impairment,

Diabetes 63 (2014) 2996–3008. https://doi.org/10.2337/db13-0970.

12. L. Chantranupong, R.L. Wolfson, J.M. Orozco, R.A. Saxton, S.M. Scaria, L. Bar-Peled,

E. Spooner, M. Isasa, S.P. Gygi, D.M. Sabatini, The Sestrins interact with GATOR2 to

negatively regulate the amino-acid-sensing pathway upstream of mTORC1, Cell Rep. 9

(2014) 1–8. https://doi.org/10.1016/j.celrep.2014.09.014.

13. N. Hatano, T. Hamada, Proteome analysis of pitcher fluid of the carnivorous plant

Nepenthes alata, J. Proteome Res. 7 (2008) 809–816. https://doi.org/10.1021/pr700566d.

14. S.D. Shumway, Y. Li, Y. Xiong, 14-3-3beta binds to and negatively regulates the tuberous

sclerosis complex 2 (TSC2) tumor suppressor gene product, tuberin, J. Biol. Chem. 278

(2003) 2089–2092. https://doi.org/10.1074/jbc.c200499200.

15. M.P. DeYoung, P. Horak, A. Sofer, D. Sgroi, L.W. Ellisen, Hypoxia regulates TSC1/2mTOR signaling and tumor suppression through REDD1-mediated 14-3-3 shuttling, Genes

Dev. 22 (2008) 239–251. https://doi.org/10.1101%2Fgad.1617608.

16. H.O. Jin, S.K. Seo, Y.S. Kim, S.H. Woo, K.H. Lee, J.Y. Yi, S.J. Lee, T.B. Choe, J.H. Lee,

S. An, S.I. Hong, I.C. Park, TXNIP potentiates Redd1-induced mTOR suppression through

stabilization of Redd1, Oncogene 30 (2011) 3792–3801.

https://doi.org/10.1038/onc.2011.102.

17. D.R. Laybutt, A.M. Preston, M.C. Akerfeldt, J.G. Kench, A.K. Busch, A.V. Biankin, T.J.

Biden, Endoplasmic reticulum stress contributes to beta cell apoptosis in type 2 diabetes.

Diabetologia 50 (2007) 752–763. https://doi.org/10.1007/s00125-006-0590-z.

18. Kaneto H, Kawamori D, Matsuoka TA, Y. Kajimoto, Y. Yamasaki, Oxidative stress and

pancreatic beta-cell dysfunction, Am. J. Ther. 12 (2005) 529–533.

https://doi.org/10.1097/01.mjt.0000178773.31525.c2.

19. C. Ebato, T. Uchida, M. Arakawa, M. Komatsu, T. Ueno, K. Komiya, K. Azuma, T.

Hirose, K. Tanaka, E. Kominami, R. Kawamori, Y. Fujitani, H. Watada, Autophagy is

important in islet homeostasis and compensatory increase of beta cell mass in response to

high-fat diet, Cell Metab. 8 (2008) 325–332. https://doi.org/10.1016/j.cmet.2008.08.009.

20. S.G. Kim, G.R. Buel, J. Blenis, Nutrient regulation of the mTOR complex 1 signaling

pathway, Mol. Cells 35 (2013) 463–473. https://doi.org/10.1007/s10059-013-0138-2.

21. T. Yuan, S. Rafizadeh, K.D.D. Gorrepati, B. Lupse, J. Oberholzer, K. Maedler, A.

Ardestani, Reciprocal regulation of mTOR complexes in pancreatic islets from humans with

type 2 diabetes, Diabetologia 60 (2017) 668–678. https://doi.org/10.1007/s00125-016-41889.

22. I.G. Chambers, P. Kumar, J. Lichtenberg, P. Wang, J. Yu, J.D. Phillips, M.A. Kane, D.

Bodine, I. Hamza, MRP5 and MRP9 play a concerted role in male reproduction and

mitochondrial function, Proc. Natl. Acad. Sci. U. S. A. 119 (2022) e211617119.

https://doi.org/10.1073/pnas.2111617119.

23. M. Andres-Alonso, M.R. Ammar, I. Butnaru, G.M. Gomes, G.A. Sanhueza, R. Raman, P.

Yuanxiang, M. Borgmeyer, J. Lopez-Rojas, S.A. Raza, N. Brice, T.J. Hausrat, T. Macharadze,

S. Diaz-Gonzalez, M. Carlton, A.V. Failla, O. Stork, M. Schweizer, E.D. Gundelfinger, M.

Kneussel, C. Spilker, A. Karpova, M.R. Kreutz, 15SIPA1L2 controls trafficking and local

signaling of TrkB-containing amphisomes at presynaptic terminals, Nat. Commun. 10 (2019)

5448. https://doi.org/10.1038/s41467-019-13224-z.

24. P.L. Garcia, Y. Liu, J. Jiricny, S.C. West, P. Janscak, Human RECQ5beta, a protein with

DNA helicase and strand-annealing activities in a single polypeptide. EMBO J. 23 (2004)

2882–2891. https://doi.org/10.1038%2Fsj.emboj.7600301.

25. K.W. Wen, B. Damania, Hsp90 and Hsp40/Erdj3 are required for the expression and antiapoptotic function of KSHV K1, Oncogene 29 (2010) 3532–3544.

https://doi.org/10.1038/onc.2010.124.

26. G.J. Ocaña, E.K. Sims, R.A. Watkins, S. Ragg, K.J. Mather, R.A. Oram, R.G. Mirmira,

L.A. DiMeglio, J.S. Blum, C. Evans-Molina, Analysis of serum Hsp90 as a potential

biomarker of β cell autoimmunity in type 1 diabetes, PLoS One 14 (2019) e0208456.

https://doi.org/10.1371/journal.pone.0208456.

27. F.F. Becker, J.D. Broome, L-asparaginase: inhibition of early mitosis in regenerating rat

liver, Science 156 (1967) 1602–1603. https://doi.org/10.1126/science.156.3782.1602.

28. L.B. Sullivan, A. Luengo, L.V. Danai, L.N. Bush, F.F. Diehl, A.M. Hosios, A.N. Lau, S.

Elmiligy, S. Malstrom, C.A. Lewis, M.G.V. Heiden, Aspartate is an endogenous metabolic

limitation for tumour growth, Nat. Cell. Biol. 20 (2018) 782–788.

https://doi.org/10.1038/s41556-018-0125-0.

29. A.S. Krall, P.J. Mullen, F. Surjono, M. Momcilovic, E.W. Schmid, C.J. Halbrook, A.

Thambundit, S.D. Mittelman, C.A. Lyssiotis, D.B. Shackelford, S.R.V. Knott, H.R. Christofk,

Asparagine couples mitochondrial respiration to ATF4 activity and tumor growth, Cell

Metab. 33 (2021) 1013–1026. https://doi.org/10.1016/j.cmet.2021.02.001.

Figure legends

Figure 1. Phosphorylation of TSC2 (T1462) and mTORC1 signaling in islets of GCN2deficient mice.

Quantitative data and representative blots from immunoblot analysis of phosphorylated

(T1462) and total TSC2 (A) and mTORC1 signaling (B, C, and D) in islets of HFD-fed wildtype mice (GCN2+/+) or HFD-fed GCN2-deficient mice (GCN2−/−). Data are presented as

means ± SEM of four independent populations from each group. *P < 0.05, **P < 0.01.

Figure 2. Search for molecules binding to 14-3-3 downstream of GCN2 signal.

(A) Hypothesis concerning the mTORC1 regulatory mechanism in pancreatic β-cells from

GCN2-deficient mice and mechanism of the pull-down assay by HaloTag. (B) Quantitative

data and representative blots in immunoblot analysis of phosphorylated (T1462) and total

TSC2 using INS-1 cells in different situations, as described below. (C) Pull-down assay of

molecules binding to 14-3-3 protein using INS-1 cells in three different situations. NC,

normal condition; AD, amino acid–deficient condition; GDAD, GCN2-deficient condition

with amino acid deficiency.

Figure 3. Quantification of candidate molecules for binding to 14-3-3 in cells and islets.

Quantitative real-time PCR analysis of each of the molecules shown in INS-1 cells under

amino acid deprivation (A), in glucose-stimulated INS-1 cells (B), in islets of HFD-fed wildtype mice (C), and in islets of HFD-fed GCN2-deficient mice (D). Quantitative real-time

PCR analysis of L-asparaginase mRNA expression in various organs (E), GCN2-knockdown

INS-1 cells (GCN2-KD) (F), and ATF4-knockdown INS-1 cells (ATF4-KD) (G) or INS-1

cells transfected with scrambled siRNA (Control). Data are presented as means ± SEM of

four independent populations from each group. *P < 0.05, **P < 0.01.

Figure 4. Analysis of mTORC1 activity in L-asparaginase-knockdown INS-1 cells.

(A) Quantitative real-time PCR analysis of L-asparaginase mRNA expression in Lasparaginase-knockdown INS-1 cells (Asparaginase-KD) or INS-1 cells transfected with

scrambled siRNA (Control). Quantitative data and representative blots in immunoblot

analysis of mTORC1 signaling (B, C, and D) in L-asparaginase-knockdown INS-1 cells

(Asparaginase-KD) or INS-1 cells transfected with scrambled siRNA (Control). (E) A model

for the regulation of mTORC1 activity by GCN2-mediated TSC2-dependent and independent pathways in pancreatic islets. Data are presented as means ± SEM of four

independent populations from each group. *P < 0.05, **P < 0.01.

Table 1. Molecules binding to 14-3-3 in INS-1 cells detected by pull-down assay under normal, amino acid–deficient, and GCN2deficient conditions under amino acid deficiency.

Normal condition

Sample #

10

11

12

13

SPROT ID

MYH7_RAT

H4_RAT

PNMA1_RAT

MYH7_RAT

PSMD1_RAT

MYH7_RAT

MYH4_RAT

PSMD1_RAT

MYPC_RAT

CLH1_RAT

PNMA1_RAT

PSMD1_RAT

PSMD1_RAT

PSMD1_RAT

GRM8_RAT

PNMA1_RAT

ATPA_RAT

PHRF1_RAT

TBB2A_RAT

TTL_RAT

TBA1A_RAT

Protein name

Myosin-7

Histone H4

Paraneoplastic antigen Ma1 homolog

Myosin-7

26S proteasome non-ATPase regulatory subunit 1

Myosin-7

Myosin-4

26S proteasome non-ATPase regulatory subunit 1

Myosin-binding protein C, cardiac-type

Clathrin heavy chain 1

Paraneoplastic antigen Ma1 homolog

26S proteasome non-ATPase regulatory subunit 1

no significant hit

26S proteasome non-ATPase regulatory subunit 1

no significant hit

26S proteasome non-ATPase regulatory subunit 1

Metabotropic glutamate receptor 8

Paraneoplastic antigen Ma1 homolog

no significant hit

ATP synthase subunit alpha, mitochondrial

PHD and RING finger domain-containing protein 1

Tubulin beta-2A chain

Tubulin--tyrosine ligase

Tubulin alpha-1A chain

14

15

16

17

18

19

20

21

22

23

24

EF1A1_RAT

PSMD1_RAT

PSMD1_RAT

G3P_RAT

1433E_RAT

PSMD1_RAT

PSMD1_RAT

PSMD1_RAT

H2A1_RAT

PSMD1_RAT

H4_RAT

PSMD1_RAT

PSMD1_RAT

Elongation factor 1-alpha 1

26S proteasome non-ATPase regulatory subunit 1

26S proteasome non-ATPase regulatory subunit 1

Glyceraldehyde-3-phosphate dehydrogenase

14-3-3 protein epsilon

26S proteasome non-ATPase regulatory subunit 1

no significant hit

no significant hit

26S proteasome non-ATPase regulatory subunit 1

26S proteasome non-ATPase regulatory subunit 1

Histone H2A type 1

26S proteasome non-ATPase regulatory subunit 1

Histone H4

26S proteasome non-ATPase regulatory subunit 1

26S proteasome non-ATPase regulatory subunit 1

SPROT ID, Swiss-Prot ID; MW, Molecular Weight

Amino acid-deficient condition

Sample #

SPROT ID

MYH6_RAT

MYH7_RAT

PSMD1_RAT

MYH7_RAT

PSMD1_RAT

MYH7_RAT

PSMD1_RAT

PSMD1_RAT

ASPG_RAT

PSMD1_RAT

H14_RAT

Protein name

Myosin-6

Myosin-7

26S proteasome non-ATPase regulatory subunit 1

Myosin-7

26S proteasome non-ATPase regulatory subunit 1

Myosin-7

26S proteasome non-ATPase regulatory subunit 1

26S proteasome non-ATPase regulatory subunit 1

N(4)-(Beta-N-acetylglucosaminyl)-L-asparaginase

26S proteasome non-ATPase regulatory subunit 1

no significant hit

Histone H1.4

10

11

12

13

14

15

16

17

18

19

20

21

22

23

HS90B_RAT

PSMD1_RAT

SI1L2_RAT

PSMD1_RAT

PSMD1_RAT

ELMD3_RAT

RECQ5_RAT

PSMD1_RAT

PNMA1_RAT

PSMD1_RAT

TBA1A_RAT

RXFP1_RAT

H14_RAT

ATPA_RAT

PSMD1_RAT

PNMA1_RAT

1433E_RAT

PHRF1_RAT

PSMD1_RAT

IMA6_RAT

H31_RAT

PHRF1_RAT

H2B1A_RAT

H2AZ_RAT

PHRF1_RAT

H2B1A_RAT

MRP5_RAT

H4_RAT

PSMD1_RAT

PNMA1_RAT

Heat shock protein HSP 90-beta

26S proteasome non-ATPase regulatory subunit 1

Signal-induced proliferation-associated 1-like protein 2

26S proteasome non-ATPase regulatory subunit 1

26S proteasome non-ATPase regulatory subunit 1

ELMO domain-containing protein 3

ATP-dependent DNA helicase Q5

26S proteasome non-ATPase regulatory subunit 1

Paraneoplastic antigen Ma1 homolog

26S proteasome non-ATPase regulatory subunit 1

Tubulin alpha-1A chain

Relaxin receptor 1

Histone H1.4

ATP synthase subunit alpha, mitochondrial

26S proteasome non-ATPase regulatory subunit 1

no significant hit

Paraneoplastic antigen Ma1 homolog

14-3-3 protein epsilon

PHD and RING finger domain-containing protein 1

26S proteasome non-ATPase regulatory subunit 1

no significant hit

Importin subunit alpha-6

Histone H3.1

PHD and RING finger domain-containing protein 1

Histone H2B type 1-A

Histone H2A.Z

PHD and RING finger domain-containing protein 1

Histone H2B type 1-A

Multidrug resistance-associated protein 5

Histone H4

26S proteasome non-ATPase regulatory subunit 1

Paraneoplastic antigen Ma1 homolog

24

ASPG_RAT

PHRF1_RAT

PSMD1_RAT

N(4)-(Beta-N-acetylglucosaminyl)-L-asparaginase

PHD and RING finger domain-containing protein 1

26S proteasome non-ATPase regulatory subunit 1

SPROT ID, Swiss-Prot ID; MW, Molecular Weight

GCN2-deficient condition under amino acid deficiency

Sample #

10

11

12

13

SPROT ID

MYH7_RAT

MYH6_RAT

MAP1B_RAT

MYH7_RAT

MYH6_RAT

MYH6_RAT

IMA6_RAT

CAN10_RAT

CLH1_RAT

ELMD3_RAT

ELMD3_RAT

HS90B_RAT

H14_RAT

CENPW_RAT

HSP7C_RAT

GRP78_RAT

H14_RAT

ATPA_RAT

Protein name

Myosin-7

Myosin-6

Microtubule-associated protein 1B

Myosin-7

Myosin-6

Myosin-6

Importin subunit alpha-6

Calpain-10

no significant hit

Clathrin heavy chain 1

ELMO domain-containing protein 3

no significant hit

ELMO domain-containing protein 3

Heat shock protein HSP 90-beta

Histone H1.4

Centromere protein W

Heat shock cognate 71 kDa protein

78 kDa glucose-regulated protein

no significant hit

no significant hit

no significant hit

Histone H1.4

ATP synthase subunit alpha, mitochondrial

14

15

16

17

18

19

20

21

22

23

24

TBB2A_RAT

TBA1A_RAT

ATPB_RAT

EF1A1_RAT

G3P_RAT

H14_RAT

1433E_RAT

TPM1_RAT

PHRF1_RAT

RXFP1_RAT

1433Z_RAT

UBP1_RAT

RS25_RAT

H31_RAT

H2B1A_RAT

PCNP_RAT

BSCL2_RAT

H2AZ_RAT

NCBP2_RAT

PHRF1_RAT

C56D2_RAT

CENPW_RAT

H4_RAT

IMA6_RAT

Tubulin beta-2A chain

Tubulin alpha-1A chain

ATP synthase subunit beta, mitochondrial

Elongation factor 1-alpha 1

no significant hit

Glyceraldehyde-3-phosphate dehydrogenase

Histone H1.4

14-3-3 protein epsilon

Tropomyosin alpha-1 chain

PHD and RING finger domain-containing protein 1

Relaxin receptor 1

14-3-3 protein zeta/delta

Ubiquitin carboxyl-terminal hydrolase 1

no significant hit

40S ribosomal protein S25

Histone H3.1

Histone H2B type 1-A

PEST proteolytic signal-containing nuclear protein

Seipin

Histone H2A.Z

Nuclear cap-binding protein subunit 2

PHD and RING finger domain-containing protein 1

Cytochrome b561 domain-containing protein 2

Centromere protein W

Histone H4

Importin subunit alpha-6

SPROT ID, Swiss-Prot ID; MW, Molecular Weight

p‐TSC2

pTSC2/TSC2

2.5

1.5

0.5

GCN2+/+

GCN2−/−

p-TSC2

TSC2

Fig. 1A

1.5

2.5

1.5

0.5

0.5

GCN2+/+

p-S6K1

S6K1

2.5

p4EBP1/4EBP1

**

2.5

**

3.5

pS6/S6

pS6K1/S6K1

p-4EBP1

p-S6

p-S6K1

**

1.5

0.5

GCN2−/−

GCN2+/+

p-S6

GCN2+/+

GCN2−/−

GCN2−/−

p-4EBP1

S6

Fig.1 B,C,D

Production of HaloTag

Wild‐type mice

GCN2−/− mice

HFD

GCN2

14‐3‐3β

GCN2

ATF4

Formation of protein complex

ATF4

14‐3‐3β

14‐3‐3β

TSC2

mTORC1

Insulin

signaling

Maintenance of

pancreatic β‐cell mass

HaloTag

TSC2

mTORC1

Negative feedback

Insulin

signaling

Pull‐down by HaloTag

Reduction in

pancreatic β‐cell mass

Fig. 2A

1.4

1.2

0.8

0.6

p-TSC2

0.4

TSC2

0.2

Fig. 2B

NC AD GDAD

205

113

79

47

34

27

17

Fig. 2C

1.5

0.5

**

**

1.8

Relative expression

Relative expression

2.5

1.6

1.4

1.2

0.8

0.6

0.4

0.2

Fig. 3A,B

2.5

1.5

0.5

1.4

**

1.2

Relative expression

Relative expression

0.8

0.6

0.4

0.2

Fig. 3C,D

L-Asparaginase

**

Relative expression

3.5

**

2.5

1.5

**

0.5

Fig. 4A

L-Asparaginase

1.2

1.2

0.8

0.6

0.4

**

0.2

Relative expression

Relative expression

L-Asparaginase

0.8

0.6

**

0.4

0.2

Control GCN2 KD

Control ATF4 KD

Fig. 4B,C

p-S6K1

L-Asparaginase

1.2

1.8

1.4

0.8

0.6

0.4

**

pS6K1/S6K1

Relative expression

1.6

1.2

0.8

0.6

0.4

0.2

0.2

p-S6K1

S6K1

Fig. 5A,B

p-4EBP1

p-S6

1.8

**

1.6

1.2

pS6/S6

1.4

p4EBP1/4EBP1

1.4

0.8

0.6

0.4

1.2

0.8

0.6

0.4

0.2

0.2

p-S6

S6

1.6

p-4EBP1

4EBP1

Fig. 5C, D

HFD

In pancreatic β‐cell

Insulin demand

Insulin synthesis

Amino acid concentration

uncharged tRNA

GCN2−/− mice

Wild‐type mice

P GCN2

GCN2

ATF4

Aspg

Sestrin2

14‐3‐3β

TSC2

mTORC1

14‐3‐3β

ATF4

Aspg

Sestrin2

TSC2

mTORC1

Negative feedback

Insulin signaling

Maintenance of

pancreatic β‐cell mass

Insulin signaling

Reduction in

pancreatic β‐cell mass

Fig. 5E

...