(A) Liver mitochondria matrix lysates from wild type mice fed ad libitum (AL) or 40% calorie restricted (CR) for 6 months were analyzed by western blotting using antibodies to CPS1, mtHSP70 and SIRT5.
(B) CPS1 activity was measured in wild type mice fed ad libitum (AL) or 40% calorie restricted (CR) for 6 months. Error bars represent SEM. (n=4 for each groups).
(C) Liver mitochondria matrix lysates from wild type mice fed ad libitum (AL), after 48 hours fasting (Fast) or 40% calorie restricted (CR) for 6 months were immuno-precipitated with anti-CPS1 antibody or normal rabbit serum (NRS). Immuno-precipitates were analyzed by western blotting with anti-CPS1 and anti pan-acetylated lysine (Ac-K) antibodies.
(D) NAD and NADH concentrations in liver mitochondria from wild type mice fed ad libitum or calorie restricted were measured by the enzyme cycling method. The increase in NAD but not NADH shown is the average of four independent experiments. Error bars represent standard deviations
(E) A model for the role of SIRT5 on CPS1 regulation in liver. In response to fasting, Nampt is induced in the cytosol to increase NMN levels, thereby triggering de novo NAD synthesis and SIRT5 activation in mitochondria. Activated SIRT5 deacetylates CPS1 and up-regulates the detoxification of ammonia by the urea cycle. A similar induction likely occurs during calorie restriction.