As anconversion of essential cofactor for lipid biosynthesisand antioxidant defense, reduced nicotinamideadenine dinucleotide phosphate (NADPH) isproduced via various pathways, including the oxidativepentose phosphate...As anconversion of essential cofactor for lipid biosynthesisand antioxidant defense, reduced nicotinamideadenine dinucleotide phosphate (NADPH) isproduced via various pathways, including the oxidativepentose phosphate pathway (oxPPP) and themalic enzyme 1 (ME1)-catalyzed conversion of malateto pyruvate. Live-cell detection of NADPH productionroutes remains challenging. Here, we reporttracing hydrides into lipid droplets (THILD), achemical imaging strategy for the detection ofpathway-specific NADPH generation in live cells. Thisstrategy exploits deuterium (2H)-labeled glucose([2H]Glc) tracers that transfer deuterides to NADPHvia specific pathways. The NADP^(2)H, in turn, transfersdeuterides to lipids, resulting in accumulation of C-2Hbonds in lipid droplets, which can be visualized bybioorthogonal stimulated Raman scattering (SRS)microscopy. We used this concept to demonstratethe imaging of oxPPP-produced NADPH using theoxPPP-specific tracer, [3-2H]Glc. Furthermore, the“switch on” of NADPH production by ME1 in differentiatingadipocytes was imaged by [4-2H]Glc. Finally,comparison of [3-2H]Glc and [4-2H]Glc THILDimaging of adipocytes showed that hypoxia inducessuppression of ME1-mediated NADPH productionand oxPPP-produced NADPH becomes the mainsource.展开更多
基金This project is supported by the National Natural Science Foundation of China(nos.91753206 and 21521003 to X.Cnos.21327808 and 21525521 to Y.H.),the National Key R&D Program of China(no.2018YFA0507600 to X.C.and no.2018YFA0108100 to Y.H.),the 2018 Beijing Brain Initiation(no.Z181100001518004 to Y.H.),the Beijing Advanced Innovation Center for Genomics(Y.H.),the US National Institutes of Health(no.R01CA163591 to J.D.R.),and the China Postdoctoral Science Foundation(no.2015M580009 to S.H.).
文摘As anconversion of essential cofactor for lipid biosynthesisand antioxidant defense, reduced nicotinamideadenine dinucleotide phosphate (NADPH) isproduced via various pathways, including the oxidativepentose phosphate pathway (oxPPP) and themalic enzyme 1 (ME1)-catalyzed conversion of malateto pyruvate. Live-cell detection of NADPH productionroutes remains challenging. Here, we reporttracing hydrides into lipid droplets (THILD), achemical imaging strategy for the detection ofpathway-specific NADPH generation in live cells. Thisstrategy exploits deuterium (2H)-labeled glucose([2H]Glc) tracers that transfer deuterides to NADPHvia specific pathways. The NADP^(2)H, in turn, transfersdeuterides to lipids, resulting in accumulation of C-2Hbonds in lipid droplets, which can be visualized bybioorthogonal stimulated Raman scattering (SRS)microscopy. We used this concept to demonstratethe imaging of oxPPP-produced NADPH using theoxPPP-specific tracer, [3-2H]Glc. Furthermore, the“switch on” of NADPH production by ME1 in differentiatingadipocytes was imaged by [4-2H]Glc. Finally,comparison of [3-2H]Glc and [4-2H]Glc THILDimaging of adipocytes showed that hypoxia inducessuppression of ME1-mediated NADPH productionand oxPPP-produced NADPH becomes the mainsource.
