Introduction
Sirtuin 1 (SIRT1) protein, the mammalian homolog for yeast silent information regulator 2 (SIR2), is a NAD+-dependent deacetylase that has emerged as a key regulator of energy metabolism (1). SIRT1 and its orthologs have been implicated in the regulation of longevity in different organisms, including yeast, Caenorhabditis elegans, Drosophila melanogaster, and mice (2, 3). In mammals, SIRT1 activation promotes improvement in diseases of aging and in features of metabolic syndrome, including the development of liver steatosis (4). Moreover, liver SIRT1 activity is decreased during experimental liver steatosis (5), and SIRT1 activation prevents the development of both nonalcoholic and alcoholic-induced liver steatosis in mice (4–7).

SIRT1 deacetylates and regulates multiple substrates, including PGC-1α (8), p53 (9), forkhead transcription factors (10), NF-κB (11), Ku70 (12), MyoD (13), and histones (14, 15). Furthermore, SIRT1 activation influences gene silencing, apoptosis, stress resistance, senescence, circadian cycles, and fat and glucose metabolism (3, 8, 16–26). Although the cellular functions of SIRT1 have been extensively investigated, less is known about the mechanisms that regulate SIRT1 activity.

SIRT1 activity can be modulated by the metabolic state of the organism, with an increase in activity during starvation and a decrease with high-caloric diets (3, 4, 19, 20, 22, 25, 27). However, how changes in SIRT1 activity are achieved during different metabolic conditions remains unclear. Several studies suggest that SIRT1 may be regulated at the transcriptional level by FOXO3a (28), p53 (28), HIC1 (29), or E2F1 (30). SIRT1 was also shown to be regulated posttranscriptionally by mRNA stabilization (31), and posttranslationally by SUMOylation (32) and phosphorylation (33). Finally, SIRT1 activity may also be influenced by changes in cellular NAD+ levels (8, 34), which are controlled by the enzymes nicotinamide phosphoribosyltransferase (NAMPT) and CD38 (35–37). Recently, we and others have demonstrated that SIRT1 activity is also modulated by protein-protein interaction (38–40) through the association of SIRT1 with the deleted in breast cancer–1 (DBC1) protein.

DBC1, localized in the cell nucleus, was initially described as being absent in certain human breast cancers. DBC1 binds directly to the catalytic domain of SIRT1, preventing substrate binding to SIRT1 and inhibiting SIRT1 activity (39, 40). However, how the DBC1-SIRT1 interaction is modulated and whether DBC1 is a regulator of SIRT1 at the physiological level in animals has not yet been determined. In the present study, we investigated the role of DBC1 as a regulator of SIRT1 during different metabolic conditions. We showed that DBC1-SIRT1 interaction was regulated during high-fat diet (HFD) feeding and during starvation, which suggests that changes in SIRT1 activity observed during different metabolic conditions could be explained by changes in DBC1-SIRT1 association. Furthermore, using our recently developed Dbc1 KO mouse model, we investigated the role of DBC1 in hepatic metabolism and the development of experimental liver steatosis induced by HFD. We found that Dbc1 KO led to both activation of hepatic SIRT1 and protection against the development of experimental liver steatosis and inflammation. Furthermore, we observed that the beneficial effect of deletion of DBC1 on the development of cellular lipid accumulation was mediated by a SIRT1-dependent mechanism. These data indicate that disruption of the DBC1-SIRT interaction may serve as a new target for the development of therapies against liver steatosis and other components of the metabolic syndrome.

Results
SIRT1 protein and NAD+ levels do not change in the liver during starvation and HFD. Several researchers have proposed that changes in SIRT1 activity observed during different metabolic conditions are primarily regulated by its own expression or by changes in NAD+ concentration inside the cell (8, 16, 34, 41). However, at least in the liver, the published data regarding these changes are contradictory (8, 27, 34). We therefore analyzed the levels of these molecules in mouse liver under different metabolic conditions. Levels of SIRT1 protein (Figure 1, A and B) and NAD+ did not vary either after 24 hours of starvation (Figure 1C, P = 0.37) or after 4 weeks of HFD (Figure 1D, P = 0.42). To further characterize NAD+ metabolism in our samples, we analyzed the expression level of 2 of the main enzymes involved in the synthesis and degradation of NAD+ in cells, namely, NAMPT and CD38 (35–37, 42, 43). Similar to our observations for NAD+, CD38 and NAMPT protein levels remained unchanged under starvation and after 4 weeks of HFD (Figure 1, A and B). Furthermore, hepatic CD38 (NADase) activity did not vary between these 2 conditions (starvation, P = 0.32; HFD, P = 0.37; Figure 1, E and F). These results demonstrate that under our experimental conditions, levels of NAD+, SIRT1, and the 2 main NAD+ metabolizing enzymes, NAMPT and CD38, remained unchanged under different metabolic conditions. These findings suggest that although NAD+ is essential for SIRT1 activity, it may not be the only factor that controls SIRT1 activity in the liver.