In lower organisms, sirtuins function to extend life span. Because of their unique NAD requirement for activity, it was suggested that sirtuins respond to food limitation to program organisms for survival during dietary stress. Indeed, SIRT1 deacetylates regulators of numerous key metabolic pathways and may thus play an important role in the adaptation to calorie restriction. Here we study a novel sirtuin SIRT5, for which no functional information has been available. We show that SIRT5 is found in the mitochondrial matrix and deacetylates a specific mitochondrial enzyme, CPS1. This enzyme mediates the first and regulated step of the urea cycle, thereby linking SIRT5 to the major pathway of ammonia detoxification and disposal in organisms. Indeed, fasted SIRT5 KO mice become hyper-ammonemic. Since the need for ammonia disposal becomes acute during fasting, our findings provide a novel example of the importance of SIRT5 in adapting to food limitation.
Regulation of ammonia disposal by SIRT5
By linking SIRT5 to beads, we identified interacting proteins from mitochondrial extracts of murine livers. The most prominent of these was carbamoyl phosphate synthetase 1 (CPS1). CPS1 catalyzes the condensation of ammonia with HCO3−
and ATP by generating carbamoyl phosphate, which is ultimately converted to urea, a molecule readily disposed from the body (Haussinger, 1990
; Meijer et al., 1990
). The urea cycle comes into play especially during fasting, when amino acids are catabolized for energy in the muscle to generate an excess of ammonia, which must be detoxified (Schimke, 1962b
The interaction between SIRT5 and CPS1 is physiologically significant by several criteria. First, the proteins interact at endogenous levels in cells. Second, SIRT5 deacetylates and activates CPS1 efficiently in vitro, while not deacetylating the canonical p53-related SIRT1 substrate. Third, SIRT5 deacetylates CPS1 in vivo; prolonged starvation triggers deacetylation of CPS1 in wild type but not SIRT5 KO mice. Fourth, SIRT5 activates and regulates CPS1 enzymatic activity in vivo and in vitro; the activity of CPS1 in ad libitum fed animals was reduced in SIRT5 KO livers, and the normal up-regulation of activity by starvation did not occur in SIRT5 KO mice. Finally, the deacetylation and activation of CPS1 was specific to SIRT5 and not other mitochondrial sirtuins. Indeed, the other mitochondrial deacetylase SIRT3 did not affect the acetylation or activity of CPS1 in vivo or in vitro.
SIRT5 appears to regulate the urea cycle in a physiologically meaningful way, since the defect in CPS1 up-regulation during starvation of SIRT5 KO mice triggers hyper-ammonemia in blood. This defect in ammonia disposal is likely specific to CPS1, since the second enzymatic step of the urea cycle, matrix OTC, is not affected by SIRT5 and the remainder of the pathway lies in the cytoplasm.
Another condition in which the urea cycle becomes very important is a high protein diet (HPD) (Schimke, 1962a
). Indeed, we observed deacetylation of CPS1 by SIRT5 in HPD mice, as well as a second level of regulation -- an increase in CPS1 protein levels. The up-regulation of CPS1 expression observed in the high protein condition may not occur during starvation, because energy for new synthesis is limited during starvation and activation by a post-translational mechanism may be most parsimonious. It will be interesting to see if high protein diets in humans, like the Atkins diet, trigger SIRT5-mediated activation of CPS1, increases in CPS1 protein levels, or both.
Mechanism of SIRT5 activation by starvation
SIRT5 protein levels do not change during starvation. However, we observed a doubling of NAD levels in liver mitochondria, with no change in NADH levels. Since we observe a steep activation of SIRT5 by NAD in vitro, we propose that this increase in NAD activates SIRT5 in the starved liver. Since NAD does not cross the mitochondrial membrane, the dramatic increase in mitochondrial NAD during starvation must be due to new synthesis (Yang et al., 2007
). Indeed, we observed induction of Nampt, the enzyme that synthesizes the NAD precursor NMN, in the starved liver cytosol. Since NMN can be imported into mitochondria and then converted into NAD (Barile et al., 1996
), we propose that starvation induces the urea cycle by increasing synthesis of NMN, which leads to de novo
synthesis of NAD in mitochondria, SIRT5 activation, and CPS1 deacetylation ().
Finally, long-term calorie restriction also gave rise to a significant increase in mitochondrial NAD levels without affecting NADH. Since this regimen also induced the deacetylation and activation of CPS1, we conclude that calorie restriction also triggers SIRT5 to up-regulate the urea cycle for ammonia disposal.
SIRT5 and SIRT4 in mitochondrial metabolism
SIRT4 represses another enzyme used during catabolism of amino acids, GDH, in this case by ADP-ribosylating it (Haigis et al., 2006
). This repression is alleviated during calorie restriction, during which there is a significant lowering in SIRT4 protein levels in liver. We show here that unlike CPS1, GDH activity is not elevated during prolonged fasting (Fig. S6, S8 and S13
). These results indicate that the level of GDH activity is not limiting in the adaptation to fasting. Since GDH is repressed both in fed and fasted mice, SIRT4, unlike SIRT5, must already be active in fed animals. We surmise that the lower level of NAD in fed mitochondria is sufficient to activate SIRT4, but not SIRT5. It will be of interest to determine whether, indeed, SIRT4 has a lower Km
for NAD than does SIRT5.
In addition, the activation of CPS1 during fasting occurred normally in mice missing either SIRT4 or the other mitochondrial sirtuin, SIRT3. Thus SIRT5 is the only sirtuin regulating the urea cycle, and that the activities of SIRT4 on GDH and SIRT5 on CPS1 appear to be surprisingly uncoupled.
Substrate specificity of SIRT5 versus SIRT1
Previous studies on the enzymatic activity of sirtuins revealed, at best, a feeble deacetylase activity for SIRT5. The robust activity we observe in the case of CPS1 indicates a high level of substrate specificity of this deacetylase. In fact, SIRT5 was completely inactive on the SIRT1 p53-related substrate, while SIRT1 was inactive on CPS1. Since SIRT5 contains only very short amino and carboxyl terminal sequences flanking the conserved, catalytic sirtuin core, it is likely that residues within the conserved domain can dictate sirtuin substrate specificity. It would be of interest to create SIRT1-SIRT5 chimeric enzymes to pinpoint the basis of sirtuin substrate specificity.
Our findings indicate another important link between sirtuins and the adaptation to food scarcity. At present, CPS1 is the only bona fide SIRT5 substrate, although other substrates may emerge. Since SIRT5 appears keyed to the conditional use of amino acids as energy sources, it is possible that other methods may reveal additional SIRT5 mitochondrial substrates involved in energy production, for example enzymes of fatty acid oxidation. Our findings strengthen the ideas that sirtuins promote metabolic adaptations during dietary changes, and that small molecule sirtuin modulators will be an important approach to treat metabolic disorders.