Fig. 6: The hyperglycemic shift from apoptosis to necroptosis depends on cellular metabolism and ROS.

a Hyperglycemic enhancement of TNF/CHX-induced death of U937 cells is prevented by inhibition of glycolysis with 2-deoxyglucose (DG). Addition of exogenous pyruvate (pyruv) restores cell death in those cells that received 2-deoxyglucose. b Annexin/PI staining and flow cytometry of U937 cells undergoing TNF/CHX-induced death at 10 and 50 mM glucose. The increase in annexin/PI staining at 50 mM glucose is prevented by 2-deoxyglucose and restored by pyruvate. Two-way ANOVA, ***p < 0.001. c High glucose causes an increase in total protein levels and phosphorylation levels of RIP1 following TNF/CHX treatment. Both total levels of RIP1 and phosphorylation levels (p-RIP1) are prevented by inhibition of glycolysis with 2-deoxyglucose. Addition of exogenous pyruvate (pyr) restores protein and phosphorylation levels of RIP1. d Flow cytometry of mitoSOX staining in U937 cells stimulated to undergo TNF/CHX-induced death. MitoSOX staining is greatly increased in high glucose conditions (50 mM). e Hyperglycemic shift to necroptosis is prevented by the antioxidant, N-acetylcysteine (NAC). f, g ELISAs measuring levels of methylglyoxal (involved in AGE formation) and total AGEs in U937 cells stimulated to undergo TNF/CHX-induced death. Levels of methylglyoxal and AGEs do not change in high glucose. h Hyperglycemic shift to necroptosis is partially prevented by inhibition of AGEs with pyridoxamine. i The hyperglycemic shift to necroptosis is not affected by inhibition of acid sphingomyelinase with desipramine (DPA). Two-way ANOVA, **p < 0.01, ***p < 0.001