Hexokinase-II positively regulates glucose starvation-induced autophagy through TORC1 inhibition - PubMed (original) (raw)

Hexokinase-II positively regulates glucose starvation-induced autophagy through TORC1 inhibition

David J Roberts et al. Mol Cell. 2014.

Abstract

Hexokinase-II (HK-II) catalyzes the first step of glycolysis and also functions as a protective molecule; however, its role in protective autophagy has not been determined. Results showed that inhibition of HK-II diminished, while overexpression of HK-II potentiated, autophagy induced by glucose deprivation in cardiomyocyte and noncardiomyocyte cells. Immunoprecipitation studies revealed that HK-II binds to and inhibits the autophagy suppressor, mTOR complex 1 (TORC1), and that this binding was increased by glucose deprivation. The TOS motif, a scaffold sequence responsible for binding TORC1 substrates, is present in HK-II, and mutating it blocked its ability to bind to TORC1 and regulate protective autophagy. The transition from glycolysis to autophagy appears to be regulated by a decrease in glucose-6 phosphate. We suggest that HK-II binds TORC1 as a decoy substrate and provides a previously unrecognized mechanism for switching cells from a metabolic economy, based on plentiful energy, to one of conservation, under starvation.

Copyright © 2014 Elsevier Inc. All rights reserved.

PubMed Disclaimer

Figures

Figure 1

Figure 1. Glucose deprivation induced autophagy is inhibited by 2-deoxy-D-glucose (2-DG) in neonatal rat ventricular myocytes (NRVMs)

(A) Cardiomyocytes were cultured in DMEM or no-glucose (NG) DMEM in the presence or absence of 2-deoxy-D-glucose (2-DG; 0.5 mM) for 16 hrs and subjected to Western blotting for LC3 (left panels). The time-course of the LC3-II/LC3-I ratio induced by glucose deprivation, plus or minus 2-DG (0.5 mM), assessed by Western blotting (right panel; n=8-10). *P<0.05, **P<0.01 vs each time point after glucose deprivation. (B) To visualize formation of autophagy, NRVMs were infected with LC3-GFP adenovirus. After 24 hrs, cells were subjected to glucose deprivation (NG) in the presence or absence of 2-DG (0.5 mM) for 16 hrs. (C) Western blotting for p62. Cells were cultured in DMEM or no-glucose (NG) DMEM in the presence or absence of 2-deoxy-D-glucose (2-DG; 0.5 mM) for 16 hrs. **P<0.01 (n=6). (D) Cardiomyocytes were cultured in DMEM or NG-DMEM in the presence or absence of 2-DG (0.1, 0.5 and 1 mM). To energize mitochondria, 5 mM pyruvate was added (lower blots). **P<0.01, ***P<0.001 n=7. Data are mean ± SEM.

Figure 2

Figure 2. Hexokinase-II (HK-II) regulates glucose deprivation induced autophagy in cardiomyocytes

(A) NRVMs transfected with control siRNA (si-Ctrl) or two different HK-II siRNAs (si-HK-II #1 and #2) were cultured in DMEM or no-glucose (NG) DMEM for 16 hrs, (n=9). (B) Cells were infected with GFP (Ad-GFP; control) or wild-type HK-II (AdHK-II) adenoviruses and subjected to 16 hrs glucose deprivation 24 hrs (n=9). (C) Cells were transfected with control siRNA (si-Ctrl), HK-II siRNA (si-HK-II-#1) or HK-I siRNA and cultured in DMEM or no-glucose (NG) DMEM in the presence or absence of 0.5 mM 2-DG for 16 hrs (n=6-8). *P<0.05, **P<0.01, ***P<0.001. ns; not significant. Data are mean ± SEM.

Figure 3

Figure 3. HK-II contributes to cardiomyocyte protection against glucose deprivation induced apoptosis

(A) NRVMs were cultured in DMEM or no-glucose (NG) DMEM for 24 hrs ± 10 mM 3-Methyladenine (3-MA) or 0.5 mM 2-DG. DNA fragmentation was determined by ELISA-based assay (n=5). (B) NRVMs transfected with control siRNA (si-Ctrl) or HK-II siRNA (si-HK-II) were subjected to glucose deprivation for 24 hrs (n=6). (C) NRVMs expressing GFP or HK-II were subjected to glucose deprivation for 24 hrs (n=8). *P<0.05, **P<0.01. Data are mean ± SEM. See also Figure S1.

Figure 4

Figure 4. HK-II contributes to decrease in TORC1 activity under glucose deprivation

(A) Representative Western blotting for phosphorylated p70S6K at Thr389 and phosphorylated 4E-BP1 at Thr37/46. NRVMs were cultured in DMEM (control) or in no-glucose (NG) DMEM in the presence or absence of 2-DG (0.5 mM) for 16 hrs. (B) Time-course of changes in P-p70S6K and P-4E-BP1 after glucose deprivation ± 2-DG (0.5 mM; n=6-8). *P<0.05, **P<0.01 vs. each time point after glucose deprivation. (C) Dose-dependent effect of 2-DG on inhibition of decrease in P-p70S6K induced by 16 hrs glucose deprivation. (D) Knockdown of HK-II attenuated the decrease in P-p70S6K induced by glucose deprivation. NRVMs transfected with control siRNA (si-Ctrl) or HK-II siRNAs (si-HK-II #1 and #2) were subjected to 16hrs glucose deprivation (n=7). (E) Overexpression of WT HK-II enhanced the inhibitory effect of glucose deprivation on P-p70S6K (n=8). *P<0.05, **P<0.01. Data are mean ± SEM.

Figure 5

Figure 5. Role of kinase activity of HK-II on regulation of autophagy under glucose deprivation

(A) NRVMs expressing GFP (control), HK-II (WT HK-II) or kinase-dead HK-II (KD HK-II) were cultured in the presence or absence of glucose for 16 hrs (n=8). (B) Representative Western blots and quantitative analysis of effects of WT and KD HK-II on p62 are shown (n=6). (C) Glucose deprivation induced increase in LC3-II and decrease in P-p70S6K are inhibited by 2-deoxy-D-glucose (2-DG), but not by 5-thio-glucose (5-TG). Cells were cultured in DMEM or NG DMEM in the presence or absence of 0.5 mM 2-DG or 0.5 mM 5-TG for 16 hrs. (D) Effect of KD HK-II in HK-II knocked down cells cultured in DMEM. HK-II was knocked down by siRNA (#1) followed by adenoviral infection with GFP, WT HK-II or KD HK-II (n=7). *P<0.05, **P<0.01, ***P<0.001, ns; not significant. Data are mean ± SEM.

Figure 6

Figure 6. HK-II associates with TORC1 and this is increased by glucose deprivation

(A) Association of HK-II with mTOR is sensitive to the detergent. Cells were harvested in 0.5 % NP-40 lysis buffer, 0.3% Chaps lysis buffer or 0.02% digitonin lysis buffer, subjected to mTOR immunoprecipitation and Western blotting for HK-II and raptor. (B) Knockdown of raptor decreases the association of mTOR with HK-II but not with GβL (upper panels). HK-I does not associate with mTOR (lower panels). NRVMs transfected with control siRNA or raptor siRNA were subjected to glucose deprivation for 16 hrs and mTOR immunoprecipitation was carried out. (C) Glucose deprivation increases association between HK-II and TORC1 which is inhibited by 0.5 mM 2-DG. NRVMs were subjected to glucose deprivation ± 2-DG for 16 hrs and mTOR or HK-II were immunoprecipitated. Right panels show quantitative analysis of the association of HK-II with mTOR (n=5), **P<0.01, ***P<0.01. (D) Adult mouse hearts perfused with NG DMEM show an increase in LC3-II, decrease in P-p70S6K, and increase in association between HK-II and mTOR. Adult mouse hearts were perfused in the Langendorff mode. After 1 hr perfusion with DMEM (control) or NG DMEM, hearts were homogenized in digitonin containing buffer and subjected to Western blotting or immunoprecipitation. Data are mean ± SEM.

Figure 7

Figure 7. The TOS motif in HK-II is responsible for HK-II binding to TORC1 and HK-II mediated regulation of autophagy under glucose deprivation

(A) HK-II contains an mTOR signaling motif (TOS motif) which is conserved in mouse, rat and human (FDIDI). (B) The association of HK-II with mTOR is increased by overexpression of wild-type (WT) HK-II, but not by the TOS motif mutant HK-II (F199A; FA). NRVMs expressing GFP (control), WT HK-II or F199A mutant HK-II were subjected to glucose deprivation for 16 hrs and HK-II was immunoprecipitated. (C) WT HK-II, but not the FA mutant, enhances the increase in LC3-II/LC3-I ratio induced by glucose deprivation (16 hrs; n=10). (D) WT HK-II, but not the FA mutant, enhances the decrease in p62 induced by glucose deprivation (16 hrs; n=8). (E) WT HK-II, but not F199A mutant (FA), provides cardiomyocyte protection against glucose deprivation. Cells were cultured in no-glucose DMEM for 24 hrs and DNA fragmentation was examined (n=7). (F) Glucose deprivation (16 hrs) decreases phosphorylation of ULK-1 at Ser757 and this is enhanced by WT HK-II, but not by F199A mutant (FA; n=10). (G) Clone 9 cells (hepatocyte cell line) were infected with AdGFP, AdHK-II or AdF199A HK-II (FA) and subjected to glucose deprivation for 18 hrs (n=5). (H) HEK293A cells were transfected with control siRNA (si-Ctrl) or HK-II siRNA (si-HK-II #1) and subjected to 8 hrs glucose deprivation (n=4-8). *P<0.05, **P<0.01, ***P<0.001. Data are mean ± SEM.

Comment in

References

    1. Adams JW, Sakata Y, Davis MG, Sah VP, Wang Y, Liggett SB, Chien KR, Brown JH, Dorn GW., 2nd Enhanced Galphaq signaling: a common pathway mediates cardiac hypertrophy and apoptotic heart failure. Proc Natl Acad Sci U S A. 1998;95:10140–10145. -PMC -PubMed
    1. Baines CP, Kaiser RA, Purcell NH, Blair NS, Osinska H, Hambleton MA, Brunskill EW, Sayen MR, Gottlieb RA, Dorn GW, et al. Loss of cyclophilin D reveals a critical role for mitochondrial permeability transition in cell death. Nature. 2005;434:658–662. -PubMed
    1. Bhaskar PT, Nogueira V, Patra KC, Jeon SM, Park Y, Robey RB, Hay N. mTORC1 hyperactivity inhibits serum deprivation-induced apoptosis via increased hexokinase II and GLUT1 expression, sustained Mcl-1 expression, and glycogen synthase kinase 3beta inhibition. Mol Cell Biol. 2009;29:5136–5147. -PMC -PubMed
    1. Chu CT, Ji J, Dagda RK, Jiang JF, Tyurina YY, Kapralov AA, Tyurin VA, Yanamala N, Shrivastava IH, Mohammadyani D, et al. Cardiolipin externalization to the outer mitochondrial membrane acts as an elimination signal for mitophagy in neuronal cells. Nat Cell Biol. 2013;15:1197–1205. -PMC -PubMed
    1. Colell A, Ricci JE, Tait S, Milasta S, Maurer U, Bouchier-Hayes L, Fitzgerald P, Guio-Carrion A, Waterhouse NJ, Li CW, et al. GAPDH and autophagy preserve survival after apoptotic cytochrome c release in the absence of caspase activation. Cell. 2007;129:983–997. -PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources