The critical cellular hydride donor NADPH is produced through various means, including the oxidative pentose phosphate pathway (oxPPP), folate metabolism and malic enzyme. In growing cells, it is efficient to produce NADPH via the oxPPP and folate metabolism, which also make nucleotide precursors. In nonproliferating adipocytes, a metabolic cycle involving malic enzyme holds the potential to make both NADPH and two-carbon units for fat synthesis. Recently developed deuterium (²H) tracer methods have enabled direct measurement of NADPH production by the oxPPP and folate metabolism. Here we enable tracking of NADPH production by malic enzyme with [2,2,3,3-²H]dimethyl-succinate and [4-²H]glucose. Using these tracers, we show that most NADPH in differentiating 3T3-L1 mouse adipocytes is made by malic enzyme. The associated metabolic cycle is disrupted by hypoxia, which switches the main adipocyte NADPH source to the oxPPP. Thus, ²H-labeled tracers enable dissection of NADPH production routes across cell types and environmental conditions.

关键的细胞氢化物供体NADPH是通过多种方式产生的，包括氧化戊糖磷酸途径（oxPPP），叶酸代谢和苹果酸酶。在生长中的细胞中，通过oxPPP和叶酸代谢产生NADPH也是有效的，叶酸代谢也是核苷酸的前体。在不增殖的脂肪细胞中，涉及苹果酸酶的代谢循环具有制造NADPH和用于脂肪合成的二碳单元的潜力。最近开发的氘（² H）示踪剂方法已经能够通过oxPPP和叶酸代谢直接测量NADPH的产生。在这里，我们能够跟踪苹果酸与[2,2,3,3- ² H]丁二酸琥珀酸和[4- ² ]产生的NADPHH]葡萄糖。使用这些示踪剂，我们表明分化3T3-L1小鼠脂肪细胞中的大多数NADPH是由苹果酸酶制造的。相关的代谢周期被缺氧破坏，缺氧将主要的脂肪细胞NADPH来源切换为oxPPP。因此，使用^2个H标记的示踪剂可以剖析跨细胞类型和环境条件的NADPH生产路线。