The coenzyme NAD⁺ is consumed by signalling enzymes, including poly-ADP-ribosyltransferases (PARPs) and sirtuins. Ageing is associated with a decrease in cellular NAD⁺ levels, but how cells cope with persistently decreased NAD⁺ concentrations is unclear. Here, we show that subcellular NAD⁺ pools are interconnected, with mitochondria acting as a rheostat to maintain NAD⁺ levels upon excessive consumption. To evoke chronic, compartment-specific overconsumption of NAD⁺, we engineered cell lines stably expressing PARP activity in mitochondria, the cytosol, endoplasmic reticulum or peroxisomes, resulting in a decline of cellular NAD⁺ concentrations by up to 50%. Isotope-tracer flux measurements and mathematical modelling show that the lowered NAD⁺ concentration kinetically restricts NAD⁺ consumption to maintain a balance with the NAD⁺ biosynthesis rate, which remains unchanged. Chronic NAD⁺ deficiency is well tolerated unless mitochondria are directly targeted. Mitochondria maintain NAD⁺ by import through SLC25A51 and reversibly cleave NAD⁺ to nicotinamide mononucleotide and ATP when NMNAT3 is present. Thus, these organelles can maintain an additional, virtual NAD⁺ pool. Our results are consistent with a well-tolerated ageing-related NAD⁺ decline as long as the vulnerable mitochondrial pool is not directly affected.

辅酶 NAD⁺ 被信号酶消耗，包括多聚 ADP-核糖转移酶 （PARP） 和 sirtuins。衰老与细胞 NAD⁺ 水平的降低有关，但细胞如何应对持续降低的 NAD⁺ 浓度尚不清楚。在这里，我们表明亚细胞 NAD⁺ 池是相互关联的，线粒体充当变阻器，在过量消耗时维持 NAD⁺ 水平。为了唤起 NAD ⁺ 的慢性、区室特异性过度消耗，我们设计了在线粒体、胞质溶胶、内质网或过氧化物酶体中稳定表达 PARP 活性的细胞系，导致细胞 NAD ⁺ 浓度下降高达 50%。同位素示踪剂通量测量和数学建模表明，降低的 NAD⁺ 浓度在动力学上限制了 NAD⁺ 的消耗，以保持与 NAD+ 生物合成速率的平衡，而 NAD⁺ 生物合成速率保持不变。除非直接靶向线粒体，否则慢性 NAD⁺ 缺陷耐受性良好。线粒体通过 SLC25A51 输入来维持 NAD⁺，并在 NMNAT3 存在时可逆地将 NAD⁺ 裂解为烟酰胺单核苷酸和 ATP。因此，这些细胞器可以维持一个额外的虚拟 NAD ⁺ 池。只要脆弱的线粒体库没有受到直接影响，我们的结果与耐受性良好的衰老相关 NAD⁺ 下降一致。