Sestrin Knockout Induces the Warburg Effect in Kidney and Primary Tumor Cells

Sestrin1, 2, and 3 are a family of stress-response proteins implicated in the pathogenesis of many diseases. Sestrins effect on AMP-activated kinase (AMPK) and mechanistic target of rapamycin (mTOR) affecting bioenergetics is most studied; however, how Sestrins directly affect metabolism is poorly understood. Wildtype (WT) HEK293T kidney cells were transfected with plasmids expressing individual FLAG-tagged Sestrins. FLAG-tagged Sestrins were immunoprecipitated and found to interact with Phosphofructokinase, a rate-limiting glycolytic enzyme, in a glucose-dependent manner. WT and Sestrin triple knockout (TKO) HEK293T cells were then subjected to glycolytic and mitochondrial stress tests and analyzed for metabolic flux via Seahorse analysis. TKO cells demonstrated higher glycolytic flux, higher ATP production but similar mitochondrial flux, and reduced proliferation compared to WT cells. Thereafter, WT and TKO cells were glucose-deprived or glucose-deprived followed by glucose addback and then characterized by steady-state metabolomics. TKO cells possessed elevations in glycolytic metabolites with glucose addback compared to WT cells. Similarly, WT and TKO cells were deprived of glucose followed by stable isotope tracing with [¹³C]glucose. TKO cells possessed higher abundances of glycolytic metabolites as well as elevated extracellular lactate. This effect resembles the Warburg effect; thus, WT and TKO primary tumor cells were tested for metabolic flux via Seahorse analysis to test the importance of Sestrins in cancer metabolism. TKO primary tumor cells demonstrated elevated glycolytic flux compared to WT. These data support a model wherein Sestrins are necessary to maintain metabolic flux homeostasis through a previously unknown regulatory mechanism involving interaction between Sestrins and metabolic enzymes.