Abstract
Hexokinase is the first enzyme in the glycolytic pathway catalyzing the reaction in which glucose is phosphorylated into glucose-6-phosphate. Mammals possess 4 isoforms of hexokinase; HK2 (hexokinase 2) is the predominant form in insulin-sensitive tissues such as adipocytes, as well as skeletal and cardiac muscle. In addition to its function in glucose metabolism, HK2 is associated with cardiomyocyte protection against mitochondrial-mediated apoptotic cell death; whether or not HK2 played a role in cardioprotective autophagy was yet to be discovered. However, in a recent study highlighted by a punctum in this issue of Autophagy, Roberts et al. addressed this possibility, uncovering a direct link between HK2, TORC1, and autophagy regulation.
Roberts et al., the authors of this study, initially showed that when neonatal rat ventricular myocytes are treated with the phosphorylatable ATP analog 2-deoxy-D-glucose (2-DG), autophagy activation decreases during glucose starvation, as shown by LC3-I to LC3-II conversion, formation of GFP-LC3 puncta, and SQSTM1/p62 degradation.Citation1 In order to understand if the effects observed by 2-DG treatment were related to HK2, the authors depleted or overexpressed HK2 using specific siRNA or adenovirus, respectively. Whereas the former leads to a decrease in LC3-I conversion, SQSTM1 degradation and apoptosis after glucose starvation, the latter has the opposite effect. Moreover, HK2 depletion blocks the effects previously observed by 2-DG treatment during glucose starvation, and treatment with siRNA to deplete HK1 fails to show any difference, providing evidence that the effects observed with 2-DG treatment are directly related to HK2 and not HK1.
After discarding the possibility that the HK2 effects on autophagy were AMPK- or AKT-dependent, the authors focused on the mechanistic target of rapamycin kinase complex 1 (MTORC1). MTORC1 activity was measured by assessing the phosphorylation levels of the known downstream targets RPS6KB/p70S6K and EIF4EBP1, showing that 2-DG treatment prevents the expected decrease in MTORC1 substrate phosphorylation after glucose starvation. Furthermore, HK2 depletion also prevents RPS6KB dephosphorylation, while HK2 adenoviral overexpression leads to an enhanced decrease in phosphorylation, suggesting a negative regulatory effect of HK2 over MTORC1.
The authors next sought to determine if HK2 kinase activity is involved in auto-phagy regulation. Incubation with the nonphosphorylatable substrate 5-thio-glucose leads to normal autophagy activation during glucose depletion, whereas treatment with the phosphorylatable analog 2-DG leads to the previously shown decrease in autophagy activity during glucose starvation. Thus, HK2 kinase activity is not necessary for MTORC1 inhibition and autophagy activation, but the presence of phosphorylated substrates proves to be a determinant for the switch of HK2 function between glycolysis and autophagy.
The authors then focus on analyzing a possible interaction between MTORC1 and HK2. Immunoprecipitation reveals a weak interaction between MTOR and HK2, which is enhanced after glucose starvation, and prevented by 2-DG treatment. A sequence analysis of HK2 reveals a potential TOR signaling motif (TOS), and mutation of the HK2 TOS motif leads to a decrease in association with MTOR. Moreover, the HK2 TOS mutant fails to activate autophagy or prevent apoptosis after glucose deprivation. Thus, the HK2 TOS motif is essential for interaction with MTORC1 and inhibition during glucose starvation.
While searching for a link between HK2 and cardioprotective autophagy, Roberts et al. found a direct association between glucose metabolism and autophagy activation. Glucose deprivation has been previously associated with autophagy by promoting a decrease in the ATP/AMP ratio, leading to AMPK activation and MTOR inhibition, which in turn results in dephosphorylation of the Atg1 mammalian homolog ULK1 and activation of autophagy. However, the data shown in this report present a new, more direct mechanism by which glucose starvation decreases the HK2 product glucose-6-phosphate leading to HK2 switching from a glycolytic enzyme to a MTOR inhibitor, thus activating autophagy.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Funding
This work was supported by grant GM053396 (to DJK).
References
- Roberts DJ, Tan-Sah VP, Ding EY, Smith JM, Miyamoto S. Hexokinase-II positively regulates glucose starvation-induced autophagy through TORC1 inhibition. Mol Cell 2014; 53:521-33; PMID:24462113; http://dx.doi.org/10.1016/j.molcel.2013.12.019.