Abstract
Deregulated nutrient signaling plays pivotal roles in body ageing and in diabetic complications; biochemical cascades linking energy dysmetabolism to cell damage and loss are still incompletely clarified, and novel molecular paradigms and pharmacological targets critically needed. We provide evidence that in the retrovirus-packaging cell line HEK293-T Phoenix, massive cell death in serum-free medium is remarkably prevented or attenuated by either glucose or aminoacid withdrawal, and by the glycolysis inhibitor 2-deoxy-glucose. A similar protection was also elicited by interference with mitochondrial function, clearly suggesting involvement of energy metabolism in increased cell survival. Oxidative stress did not account for nutrient toxicity on serum-starved cells. Instead, nutrient restriction was associated with reduced activity of the mTOR/S6 Kinase cascade. Moreover, pharmacological and genetic manipulation of the mTOR pathway modulated in an opposite fashion signaling to S6K/S6 and cell viability in nutrient-repleted medium. Additionally, stimulation of the AMP-activated Protein Kinase concomitantly inhibited mTOR signaling and cell death, while neither event was affected by overexpression of the NAD+ dependent deacetylase Sirt-1, another cellular sensor of nutrient scarcity. Finally, blockade of the mTOR cascade reduced hyperglycemic damage also in a more pathophysiologically relevant model, i.e. in human umbilical vein endothelial cells (HUVEC) exposed to hyperglycemia. Taken together these findings point to a key role of the mTOR/S6K cascade in cell damage by excess nutrients and scarcity of growth-factors, a condition shared by diabetes and other ageing-related pathologies.
Highlights
Mammalian cells sense availability of nutrients through a complex array of both paracrine/endocrine and cellautonomous signaling cascades which regulate proliferation, differentiation and survival
Consistent with previous reports, we found increased levels of AkT phosphorylation/activity in cells deprived of glucose or treated with Rapamycin, in a fashion which inversely correlated with the activation of the mTOR effector S6 kinase (Figure 5 A, a). mTOR-silenced cells displayed increased phosphorylation of AkT in nutrient rich medium, to a lower extent compared to control cells treated with Rapamycin (Figures 5A, a and b); transfection of rat mTOR cDNA in cells deprived of human mTOR rescued mTOR expression and activity and in parallel decreased the phosphorylation of AkT (Supplementary data, S1)
We describe here a novel mechanism for cell survival regulation by nutrients, our major conclusion being that activation of the mTOR signaling pathway is detrimental to cell survival in the context of growth factor scarcity
Summary
Mammalian cells sense availability of nutrients through a complex array of both paracrine/endocrine and cellautonomous signaling cascades which regulate proliferation, differentiation and survival. By phosphorylating and activating the S6 kinase or inhibiting the Elongation Factor 4 Binding Protein-1 (4EBP-1), mTOR stimulates the ribosomal translation of different classes of mRNAs, thereby promoting protein synthesis. It acts directly on gene expression by phosphorylating transcription factors mainly involved in the orchestration of glucose and lipid metabolism [7]. The mTOR cascade integrates nutritional and mitogenic/antiapoptotic cues ensuring that energy supply and protein synthesis are adequate to support cell growth (i.e. increase in cell size), proliferation, and accumulation of biomass
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