Figure 6. Starvation and high protein diet up-regulate CPS1 activity in vivo.

(A) Liver mitochondria matrix lysates from wild type and SIRT5 KO mice fed ad libitum or after 48 hours fasting were analyzed by western blotting using antibodies to CPS1, mtHSP70, SIRT5 and SIRT4.

(B) CPS1 activity was measured in wild type and SIRT5 KO mice fed ad libitum or after 48 hours fasting. Error bars represent SEM. (n=8 for each groups). Starvation induces CPS1 activity in wild type but not SIRT5 KO mice.

(C) Liver mitochondria matrix lysates in wild type and SIRT5 KO mice fed normal chow or a high protein diet for 2 weeks were analyzed by western blotting using antibodies to CPS1, mtHSP70, SIRT5 and SIRT4. CPS1 protein levels were induced by the high protein diet in both wild type and SIRT5 KO mice.

(D) CPS1 activity was measured in wild type and SIRT5 KO mice fed normal chow diet or high protein diet for 2 weeks. Error bars represent SEM. (n=4 for each groups). High protein diet induces CPS1 in wild type and to a lesser degree in SIRT5 KO mice.

(E) CPS1 protein levels in mitochondria matrix in wild type mice fed ad libitum or fasted for 48 hours or fed High protein diet for 2 weeks. The intensity of CPS1 band relative to mtHSP70 was analyzed by NIH ImageJ. Data were shown as % to AL control. Error bars represent SEM. (n=4 for each groups).

(F) Liver mitochondria matrix lysates from wild type and SIRT5 KO mice fed ad libitum or after 48 hours fasting 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. Fasting triggered deacetylation of CPS1 in wild type but not SIRT5 KO mice.

(G) NAD and NADH concentrations in liver mitochondria from wild type mice fed ad libitum or after 48 hr fasting 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

(H) Mice were fed ad libitum (AL) or starved 48-hours, and whole cell (upper) and mitochondrial (lower) extracts were blotted with antibodies to Nampt, actin and mtHSP70. Nampt was induced 2–3 fold by starvation in whole cell extracts (left), and was not associated with purified mitochondria (right).

(I) Blood ammonia was measured in wild type and SIRT5 KO mice fed ad libitum or after 48 hours fasting. Error bars represent SEM. (n=8 for each groups). Starvation caused hyper-ammonemia in SIRT5 but not wild type mice.