Chronic Cellular <scp>NAD</scp> Depletion Activates a Viral Infection‐Like Interferon Response Through Mitochondrial <scp>DNA</scp> Leakage

Nicotinamide adenine dinucleotide (NAD) is a key coenzyme involved in energy metabolism, DNA repair, and cellular signaling. While the effects of acute NAD depletion have been better characterized, the consequences of chronic NAD deficiency remain unclear. Here, we investigated the impact of chronic NAD depletion in cultured cells by removing the availability of nicotinamide (NAM), a key precursor for NAD synthesis, from the culture media. In NIH3T3 fibroblasts, NAM depletion caused a dramatic drop in intracellular NAD levels within 2 days. Remarkably, the cells remained viable even after 7-14 days of NAM depletion, despite NAD⁺ levels falling to less than 10% of control conditions. This chronic NAD depletion led to distinct metabolic alterations. Mitochondrial basal respiration remained unchanged, but cells exhibited reduced spare respiratory and maximal capacities, along with significantly impaired glycolysis. Notably, NAD depletion triggered an interferon-dependent inflammatory response, resembling viral infections. This was driven by cytosolic leakage of mitochondrial DNA (mtDNA) through voltage-dependent anion channel 1 (VDAC1), which activated the cGAS-STING signaling pathway. Inhibition of VDAC oligomerization with VBIT-4, STING signaling with H-151, or mtDNA depletion blocked the upregulation of interferon genes induced by NAM depletion. Similar interferon responses triggered by NAD depletion were observed in IMR90 human fibroblasts and HS5 stromal cells. Our findings reveal a novel link between chronic NAD deficiency, VDAC-mediated mtDNA release to the cytoplasm, and the activation of the inflammatory response, providing new insight into how NAD decline affects cellular metabolic and inflammatory processes.