The Rate of NAD+ Breakdown Is Maintained Constant against Deletion or Overexpression of NAD+-Degrading Enzymes in Mammalian Cells.

Abstract

Cellular NAD⁺ is continuously degraded and synthesized under resting conditions. In mammals, NAD⁺ synthesis is primarily initiated from nicotinamide (Nam) by Nam phosphoribosyltransferase, whereas poly(ADP-ribose) polymerase 1 (PARP1) and 2 (PARP2), sirtuin1 (SIRT1), CD38, and sterile alpha and TIR motif containing 1 (SARM1) are involved in NAD⁺ breakdown. Using flux analysis with ²H-labeled Nam, we found that when mammalian cells were cultured in the absence of Nam, cellular NAD⁺ levels were maintained and NAD⁺ breakdown was completely suppressed. In the presence of Nam, the rate of NAD⁺ breakdown (R_B) did not significantly change upon PARP1, PARP2, SIRT1, or SARM1 deletion, whereas stable expression of CD38 did not increase R_B. However, R_B in PARP1-deleted cells was much higher compared with that in wild-type cells, in which PARP1 activity was blocked with a selective inhibitor. In contrast, R_B in CD38-overexpressing cells in the presence of a specific CD38 inhibitor was much lower compared with that in control cells. The results indicate that PARP1 deletion upregulates the activity of other NADases, whereas CD38 expression downregulates the activity of endogenous NADases, including PARP1 and PARP2. The rate of cellular NAD⁺ breakdown and the resulting NAD⁺ concentration may be maintained at a constant level, despite changes in the NAD⁺-degrading enzyme expression, through the compensatory regulation of NADase activity.