Glucose Availability Regulates Sestrin3 Binding to Hexokinase2 to Affect Glucose‐induced mTORC1 Activation
Authors: Roberson Paul A. , Welles Jaclyn E. , Xu Dandan , Kincheloe Gregory N. , Jefferson Leonard S. , Kimball Scot R. 
Published: May 2022

The Sestrins (Sesn), a family of stress‐response proteins (Sesn1‐3), coordinate metabolism and protein synthesis by affecting Mechanistic Target of Rapamycin in Complex 1 (mTORC1) activation. Prior research demonstrates a role for Sesns in the regulation of mTORC1 by amino acids, but mechanisms regulating glucose‐induced stimulation of mTORC1 are undefined.Initially, glucose deprivation in C2C12 myotubes was found to attenuate mTORC1 activation, while glucose resupplementation thereafter stimulated mTORC1 activation. Similarly, rats that were fasted overnight and then given an oral gavage of glucose showed mTORC1 activation in the tibialis anterior muscle (TA) compared to saline‐gavaged rats.To elucidate the role of Sesns in affecting mTORC1 activation, HEK293T wild‐type (WT) and Sesn‐ablated cells (SesnTKO) were incubated in Dulbecco’s Modified Eagle Medium (DMEM) containing (CM) or lacking glucose (‐Glu) or DMEM lacking glucose followed by glucose readdition (GluAB). In WT, but not SesnTKO cells, GluAB activated mTORC1 showing that Sesns are necessary for glucose‐induced activation of mTORC1.To determine potential mechanisms, plasmids expressing FLAG‐Sesn1, 2, or 3 or a control protein were transfected into HEK293T cells. Cells were treated similarly to the above media conditions, harvested, immunoprecipitated using FLAG beads, and probed for protein interactions via western blot. Interestingly, Hexokinase2 (HK2), but not HK1, was associated with Sesn1‐3, and the interaction was more prominent in ‐Glu compared to CM and GluAB.Based on this finding and previous studies showing Sesn3 affects glucose metabolism, we hypothesized Sesn3 attenuates glucose‐induced mTORC1 activation. To characterize this interaction, we performed similar immunoprecipitations; however, in CM and ‐Glu cells, glucose was added directly to cell lysates to determine if glucose directly altered the interaction between Sesn3 and HK2. Glucose addition to lysates did not affect the interaction suggesting glucose does not directly modify the interaction.We also found that the interaction is particularly influenced by glucose as individual resupplementation of leucine, fructose, sodium pyruvate, nor mannitol following glucose deprivation dissociated the complex.To illustrate in vivo reproducibility, plasmids expressing FLAG‐Sesn3 or Green fluorescent protein (GFP) were transfected into either TA of 12 rats. Rats were then given a glucose or saline oral gavage, and then both TAs were excised and analyzed via western blot. Notably, glucose‐induced activation of mTORC1 occurred in the GFP‐leg of glucose‐gavaged animals; however, the Sesn3‐leg of glucose‐gavaged animals showed activation similar to saline‐gavaged animals suggesting Sesn3 attenuates glucose‐induced mTORC1 activation.These data demonstrate Sesn3 affects glucose‐induced stimulation of mTORC1 through a possible mechanism involving HK2. Importantly, the precise mechanism for how Sesn3 affects mTORC1 warrants further investigation.