Live-Cell Imaging of NADPH Production from Specific Pathways

As anconversion of essential cofactor for lipid bio-synthesis and antioxidant defense, reduced nicotinamide adenine dinucleotide phosphate (NADPH) is produced via various pathways, including the oxidative pentose phosphate pathway (oxPPP) and the malic enzyme 1 (ME1)-catalyzed conversion of malate to pyruvate. Live-cell detection of NADPH production routes remains challenging. Here, we report tracing hydrides into lipid droplets (THILD), a chemical imaging strategy for the detection of pathway-specific NADPH generation in live cells. This strategy exploits deuterium (2H)-labeled glucose ([2H]Glc) tracers that transfer deuterides to NADPH via specific pathways. The NADP(2)H, in turn, transfers deuterides to lipids, resulting in accumulation of C-H-2 bonds in lipid droplets, which can be visualized by bioorthogonal stimulated Raman scattering (SRS) microscopy. We used this concept to demonstrate the imaging of oxPPP-produced NADPH using the oxPPP-specific tracer, [3-H-2]Glc. Furthermore, the "switch on" of NADPH production by ME1 in differentiating adipocytes was imaged by [4-H-2]Glc. Finally, comparison of [3-2H]Glc and [4-2H]Glc THILD imaging of adipocytes showed that hypoxia induces suppression of ME1-mediated NADPH production and oxPPP-produced NADPH becomes the main source.