Akt regulation of glycolysis mediates bioenergetic stability in epithelial cells

Abstract

Cells use multiple feedback controls to regulate metabolism in response to nutrient and signaling inputs. However, feedback creates the potential for unstable network responses. We examined how concentrations of key metabolites and signaling pathways interact to maintain homeostasis in proliferating human cells, using fluorescent reporters for AMPK activity, Akt activity, and cytosolic NADH/NAD + redox. Across various conditions, including glycolytic or mitochondrial inhibition or cell proliferation, we observed distinct patterns of AMPK activity, including both stable adaptation and highly dynamic behaviors such as periodic oscillations and irregular fluctuations that indicate a failure to reach a steady state. Fluctuations in AMPK activity, Akt activity, and cytosolic NADH/NAD + redox state were temporally linked in individual cells adapting to metabolic perturbations. By monitoring single-cell dynamics in each of these contexts, we identified PI3K/Akt regulation of glycolysis as a multifaceted modulator of single-cell metabolic dynamics that is required to maintain metabolic stability in proliferating cells.

Cell-to-cell variability in AMPK activation reveals autonomous cycles in cellular energy balance

“…Activation of AMPK and its downstream effectors may be beneficial for treating diabetes, cancer, and other diseases, and there have been a number of efforts to develop pharmacological AMPK activators (Cokorinos et al, 2017;Myers et al, 2017). In particular, biguanides are an important class of diabetes drug that act in part as ETC inhibitors, and which induce cyclic AMPK activation similar to that shown here (Hung et al, 2017a). Understanding the kinetics of AMPK in response to ETCi challenges, and the factors that underlie the heterogeneous ETCi response, will be important in optimizing AMPK activation strategies for beneficial cellular effects.…”

“…However, this variation could also arise from other sources, including non-linearity or non-specificity in the reporter readout. We investigated these alternatives using the non-tumor epithelial cell line MCF10A, a system that responds with metabolic changes to physiological signals including insulin, growth factors, and attachment (Grassian et al, 2011;Hung et al, 2017a;Schafer et al, 2009). To quantitatively relate AMPKAR2 reporter signals to cellular AMPK activity, we calibrated the FRET ratio measured by microscopy to AMPKAR2 phosphorylation status using Phos-tag electrophoresis ( Figure S1A-B).…”

“…Time-lapse wide-field microscopy was performed as described previously (Hung et al, 2017b;Pargett et al, 2017). Briefly, 25000 cells were seeded 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.…”

“…Recently, using a live-cell AMPK activity reporter (AMPKAR2), we discovered a high degree of variability in AMPK activity in cultured epithelial cells (Hung et al, 2017a). Treatment with ETC inhibitors (ETCi) induced cycles of AMPK activation with a period of 3-5 hours, indicating that, at least under certain conditions, the metabolic activity of individual cells can cycle in a rhythm independent from the cell cycle.…”

Berberine ameliorates renal impairment and inhibits podocyte dysfunction by targeting the phosphatidylinositol 3‐kinase–protein kinase B pathway in diabetic rats

“…In addition, a study has shown that TGF-b1 could also activate the phosphatidylinositol 3 kinase-protein kinase B (PI3K-Akt) signaling pathway 8 . The PI3K-Akt pathway is involved in many cellular processes, including proliferation, differentiation, apoptosis, tumor growth and cell cycle progression [9][10][11] . Therefore, it is important to know whether there is a relationship among costimulation of TGF-b1 and high glucose, podocyte apoptosis and the PI3K-Akt pathway, and the details.…”