Author response: Akt regulation of glycolysis mediates bioenergetic stability in epithelial cells

Transient phases of OXPHOS inhibitor resistance reveal underlying metabolic heterogeneity in single cells

“…Can the remaining variability then be attributed to different rates of glucose uptake? This is possible, as we have observed that AMPK and AKT activity can fluctuate during normal growth conditions (Hung et al, 2017), which would be expected to result in different glucose uptake rates over time. Still another possibility is that, as in yeast (Cai and Tu, 2012;Tu et al, 2007), mammalian cells have an intrinsic cycle that controls flux through glycolysis and protein synthesis.…”

“…The reporters AMPKAR2 (Hung et al, 2017) and ERKTR-mCherry (Sparta et al, 2015) were previously described. PercevalHR (Tantama et al, 2013), ATeam1.03 (Imamura et al, 2009), and GLUT1 were obtained from Addgene.…”

“…Live-cell fluorescence microscopy Time-lapse wide-field microscopy was performed as described previously (Hung et al, 2017;Pargett et al, 2017). Briefly, 25,000 cells were plated one day prior to imaging in glass-bottom 96-well plates (Cellvis P96-1.5H-N, Mountain View, CA) pretreated with type I collagen (GIBCO A10483-01) to promote cell adherence.…”

“…Current approaches measure the expression profiles of metabolic enzymes (Hartmann et al, 2020;Xiao et al, 2019), rather than directly assessing metabolites or their flux. Other work has used biosensors to reveal variability in clonal cell lines responding to inhibitors of metabolism (Hung et al, 2017) and in cellular metabolic disposition based on responses to glucose withdrawal (Depaoli et al, 2018). However, while it is typical for single-cell approaches to reveal heterogeneity, it is not clear how the observed variation originates within an isogenic population of cells, and what impact it has on pathways downstream of the immediate response.…”

“…While AMPK is not required for all the effects of OXPHOS inhibition (Griss et al, 2015), its activity and phosphorylation status, or phosphorylation of its effectors such as acetyl-CoA carboxylase (ACC), are useful indicators of cellular energetic status. Recently, fluorescent protein-based FRET reporters have enabled tracking of AMPK activity in living cells, revealing the localization and kinetics of its activity in response to different forms of metabolic perturbation, including OXPHOS inhibition (Hung et al, 2017;Konagaya et al, 2017;Tsou et al, 2011).…”