Sirtinol promotes PEPCK1 degradation and inhibits gluconeogenesis by inhibiting deacetylase SIRT2

Phosphoenolpyruvate carboxykinase 1 (PEPCK1) is the critical enzyme for gluconeogenesis and is linked with type II diabetes. Previous studies have found that SIRT2, a deacetylase, plays an important role in deacetylating PEPCK1 and little is known about the anti-diabetic activity of SIRT2 inhibitors. In this study, we investigated the anti-diabetic effects of sirtinol, a SIRT2 inhibitor, on cell gluconeogenesis in vivo and in vitro. Immunoblotting analysis revealed that sirtinol significantly decreased the protein level of PEPCK1, and was accompanied by the hyperacetylation of PEPCK1 as well as decreased glucose output in a dose-dependent manner. Furthermore, sirtinol exerted little impact on endogenous PEPCK1 levels in SIRT2-knockdown cells. The in vitro experiments further confirmed the in vivo data; sirtinol decreased liver PEPCK1 protein level and prevented pyruvate-induced blood glucose from increasing. Based on our results, the rate-limiting enzyme PEPCK1 is the primary target of sirtinol, and the inhibition of SIRT2 activity may play an important role in cell gluconeogenesis. Thus, SIRT2 may be a novel molecular target for diabetes therapy and may thus shed light on the underlying diabetes treatment mechanisms of sirtinol.

from lysine residues and is accompanied with hydrolysis of NAD to generate nicotinamide (NAM), lysine, and O-acetyl-ADP-ribose. Consequently, NAM can be serve as an inhibitor to this kind of enzymatic reaction 11,[14][15][16] . SIRT2 is primarily a cytoplasmic protein, and tubulin 17 as well as PEPCK1 3 are well known substrates of this deacetylase. As a SIRT2 inhibitor, sirtinol has been shown to have anti-tumor [18][19][20][21][22] and anti-inflammatory 23,24 properties, but its impact on metabolism as well as its molecular mechanisms of action have not yet been reported.
In the present study, we focused on the anti-gluconeogenesis effect of sirtinol and explored its molecular mechanisms. We discovered that sirtinol-induced acetylation plays a critical role in protein post-translational modification of PEPCK1 and cell gluconeogenesis by targeting SIRT2. Additionally, the hypoglycemic effects of sirtinol on glucose output and gluconeogenesis were confirmed in vitro as well as in vivo.

Results
Sirtinol increases the acetylation of PEPCK1 by inhibiting the activity of SIRT2. Since Lys70, Lys71, and Lys594 were identified as key acetylated sites of human PEPCK1 and these are deacetylated by SIRT2 3,25 , we first synthesized a peptide containing two acetylated sites (Lys70, Lys71) of PEPCK1 and incubated it with prokaryotic purified SIRT2 protein. We then assessed peptides by mass spectrometry (MS) (Fig. 1A). As expected, the deacetylation effect of SIRT2 was abolished by sirtinol in dose-dependent manner. Next, using a eukaryotic purified SIRT2 deacetylated system (Fig. 1B), we found that Lys40-acetylation levels of tubulin and overall-acetylation levels of PEPCK1 were significantly reduced following SIRT2 and its coenzyme treatment. However, this effect was eliminated after treating with either NAM or sirtinol, resulting in hyperacetylation of tubulin and PEPCK1 (Fig. 1C). We further studied the inhibitory effect of sirtinol in HEK293T cells, and consistent with previous deacetylation results, the deacetylation effect of SIRT2 was abolished after treatment with either NAM or sirtinol (Fig. 1D). As His187 is the key conserved site controlling the activity of SIRT2, mutant SIRT2 or inhibitor treatment caused no significant change to the acetylation of target proteins compared to the wild type of SIRT2 (Fig. 1D). Taken together, these results suggest that sirtinol increases the acetylation of PEPCK1 by inhibiting the activity of SIRT2 specifically.
Sirtinol increases the acetylation of PEPCK1 at three key acetylation sites. Acetylation of three key acetylation sites of PEPCK1 (Lys70, Lys71 and Lys594) leads to destabilized proteins, accompanied by decreased levels of PEPCK1 25 . We further explored whether the increased overall-acetylation of PEPCK1 caused by sirtinol is associated with hyperacetylation of these key sites. HEK293T cells were overexpressed with flag-tagged PEPCK1 then treated as indicated. Sirtinol increased the overall-acetylation levels of PEPCK1 significantly in dose-dependent manner ( Fig. 2A). However, the hyperacetylation inducing effect of sirtinol was abolished after the lysines (K) of three acetylation sites of PEPCK1 mutated to arginines (R) or glutamine (Q), resulting in marginal acetylation change of PEPCK1 as compared to the untreated group (Fig. 2B). Hyperacetylation of three key acetylation sites of PEPCK1 resulted its decreased protein levels 25 . As expected, SIRT2 overexpressed hepatocytes had an increased PEPCK1 level as compared to hepatocytes, which transfected with control plasmid. This effect was attenuated following treatment with sirtinol ( Fig. 2C and D). These results suggest that hyperacetylation caused by sirtinol is dominantly due to acetylation of Lys70, Lys71 and Lys594 of PEPCK1.

Sirtinol decreases protein levels of PEPCK1 and cell gluconeogenesis. Sirtinol exhibits an
anti-proliferation effect to certain tumor cells [18][19][20][21][22] . Consequently, we assessed the cytotoxicity of sirtinol in HEK93T cells by the CCK-8 assay. HEK293T cells showed a tolerance to sirtinol even under as much as 200 μM sirtinol treatment (Fig. 3A). Using HEK293T and mouse hepatocytes, we found that sirtinol significantly reduced the PEPCK1 levels in a concentration-and time-dependent manner without changing its mRNA level (Fig. 3B, C and D). Since PEPCK1 plays an important role in gluconeogenesis and catalyzes its rate-limiting step 6-9 , we further explored cell gluconeogenesis capability. As expected, cell gluconeogenesis capability was closely related to protein levels of PEPCK1. Sirtinol significantly reduced the cell glucose output in a dose-dependent manner and gluconeogenesis decreased as much as 30% under 50 μM sirtinol treatment for 4 hours ( Fig. 3D and E). Interestingly, sirtinol treatment did not degrade acetylation site mutated PEPCK1 proteins (SFig. A and B).
Sirtinol-induced acetylation promotes PEPCK1 degradation via ubiquitin-proteasome pathway. In order to elucidate how sirtinol-induced hyperacetylation of PEPCK1 promotes PEPCK1 degradation, primary mouse hepatocytes were each treated with sirtuins deacetylase inhibitor nicotinamide (NAM), a chemical that supposedly inhibits all Class III deacetylases 26 . In confirmation with our previous finding 25 , levels of endogenous PEPCK1 were decreased significantly (Fig. 4A). Accordingly, the PEPCK1 decreasing effect of sirtinol can be abolished by the overdose of the deacetylase inhibitor (Fig. 4A). Next, we inhibited PEPCK1 synthesis by using CHX (Cycloheximide) and found that PEPCK1 degraded faster in presence of sirtinol (Fig. 4B). Furthermore, when MG132 was included to inhibit proteasomal degradation, sirtinol-induced PEPCK1 degradation was blocked (Fig. 4C). These results suggest that the ubiquitin-proteasome pathway mediates acetylation-promoted decrease of PEPCK1. Consistent with these results, active PEPCK1 ubiquitinylation was detected and sirtinol significantly increased PEPCK1 ubiquitinylation (Fig. 4D) after Flag-tagged PEPCK1 and HA-tagged ubiquitin were coexpressed in HEK293T cells,. However, the role of sirtinol as promoter of PEPCK1 ubiquitination was not observed after lysines (K) on three acetylation sites of PEPCK1 mutated to arginines (R) or glutamine (Q) (Fig. 4E).
Sirtinol destabilizes PEPCK1 and inhibits gluconeogenesis by targeting on SIRT2. As a NAD + -dependent class III deacetylase, SIRT2 deacetylates and stabilizes PEPCK1 25 . We investigated whether SIRT2 is primarily responsible for decreasing protein levels of PEPCK1 and cell gluconeogenesis of sirtinol. We found that knockdown of SIRT2 by shRNA significantly decreased endogenous PEPCK1 levels and blocked the Scientific RepoRts | 7: 7 | DOI:10.1038/s41598-017-00035-9 PEPCK1 decreasing effect of sirtinol (Fig. 5A). Furthermore, when wild-type SIRT2, but not its catalytically inactive H187Y mutant 27,28 , was put back in SIRT2-knockdown 293T cells, a significant increase of PEPCK1 protein levels was observed. Moreover, sirtinol reversed the PEPCK1 increasing effect of wild-type SIRT2 (Fig. 5B). Hepatocytes from Sirt2 −/− mice had a decreased endogenous PEPCK1 level compared to their counterparts, and Sirt2 knock out also blocked the PEPCK1 decreasing effect of sirtinol (Fig. 5C), consistent with SIRT2-knockdown results from 293T cells. This validated our model that acetylation destabilizes PEPCK1 and that PEPCK1 protein levels are closely related to the amount of gluconeogenesis, which is regulated by sirtinol (Fig. 5D). In addition, since sirtinol has inhibitory effect on SIRT1, we further explored whether the inhibitor or agonist of SIRT1 could influence the protein levels of PEPCK1 and found that SIRT1 did not affect the protein levels of PEPCK1 (SFig. C).
Sirtinol destabilizes PEPCK1 and inhibits gluconeogenesis in mice. Finally, we investigated whether PEPCK1 level and gluconeogenic rate could be regulated by injecting sirtinol in mice. Since pyruvate Specified reactions were incubated with 5 mM nicotinamide or 50 μM sirtinol. One half of reaction products were visualized by Western blotting with specific antibody (up). The other half of the immunoprecipitated material was incubated with PEPCK1 antibody and protein A agarose then washed and visualized by Western blotting with antibody for pan-acetylated protein and PEPCK1 (down). (D) HEK293T cells were transfected with flag tagged wild type or mutant SIRT2 (H187Y) plasmid for 36 hours, then treated with indicated 5 mM NAM or 50 μM sirtinol for 4 hours. Cells were harvested and lysated. One half of lysates were visualized by Western blotting with specific antibody (up). The other half of the lysates were immunoprecipitated with flagbeads, then washed and visualized by Western blotting with antibody for pan-acetylated protein and PEPCK1 (down). is a source for gluconeogenesis, we employed pyruvate tolerance tests. At 1 hour after intraperitoneal injection, sirtinol effectively reduced blood glucose levels by approximately 40% compared to the control group in mice (Fig. 6A). Simultaneously, sirtinol decreased PEPCK1 protein levels in mice liver (Fig. 6B), and Sirt2 −/− mice displayed low gluconeogenic ability. Furthermore, sirtinol could not decrease PEPCK1 or blood glucose ( Fig. 6C and D). This was consistent with our in vitro findings. Together, these results demonstrate that sirtinol-induced PEPCK1 destabilization may serve as an important mechanism to regulate the rate of gluconeogenesis.

Discussion
Biological anti-diabetic strategies have been widely developed in the past few decades. Nevertheless, new chemical anti-diabetic drugs are so few that the clinical outcome for advanced diabetic patients remains dependent on insulin injections. Growing evidence suggests that acetylation is a conservative protein modification and that regulation of glucose metabolism enzymes may affect gluconeogenesis. This provides a new strategy to develop anti-diabetic drugs by regulating protein acetylation 2,3,25 . In this study, we have uncovered that sirtinol, a SIRT2 inhibitor, destabilized the metabolic enzyme PEPCK1 and decreased gluconeogenesis in vitro and in vivo.
Sirtinol selectively inhibits NAD + -dependent deacetylases SIRT1 and SIRT2, as it shares the β-naphthol pharmacophore with other competitive sirtuin inhibitors, such as cambinol and splitomicin 29 . SIRT1, one of the most widely reported members of the SIRT family, is known to regulate cell proliferation, apoptosis, and migration 13,30 . Targeting SIRT1, sirtinol-caused hyperacetylation of p53, Ku70, and FOXO3a, as well as phosphorylation of MAPK, leads to significant cytotoxic effect on breast and colon cancer cells 21,22 . However, our cell viability test and mouse pyruvate tolerance test demonstrated that sirtinol was well tolerated in the absence of other noxious stimuli. These may be due to less malignant cells and in absence of stress stimuli (e.g. cell starvation, sirtinol overdose, and chemotherapeutic agents) in our study.
SIRT2, broadly conserved through organisms ranging from bacteria to humans, catalyzes a wide range of biological processes including genetic control, development, and metabolism, in which the removal of the acetyl group from lysine residues is coupled with the hydrolysis of NAD to generate nicotinamide, lysine, and O-acetyl-ADP-ribose 11, 14-16 . There are many SIRT2 inhibitors that are generally classified into different groups: nicotinamide, as a substance that inhibits NAD + -dependent reactions in general 31,32 ; and SIRT2-specific inhibitors, such as sirtinol 33 , spitomicin 34 and cambinol 18 . A well-studied substrate of SIRT2 is α-tubulin. SIRT2 deacetylates lysine-40 of α-tubulin both in vitro and in vivo, and the acetylation of α-tubulin was used as an indicator of SIRT2 activity in our study 17 . PEPCK1 is another substrate of SIRT2. In both yeast and humans, acetylation of PEPCK1 has been reported, and its catalytic activity is inactivated following acetylation 3, 10 . Lys70, Lys71, and Lys594 of human PEPCK1 was found to be acetylated, and acetylation of these sites led to decreased protein stability, reduced protein levels, and declined gluconeogenesis without affecting mRNA levels 3 . Consistent with previous studies, we found that inhibition of the SIRT family increased acetylation of PEPCK1, as well as destabilized it by inhibiting SIRT2 activity. However, compared with SIRT1-mediated translational control that usually takes hours to occur, this SIRT2-mediated post-translational modification control on PEPCK1 by sirtinol only took minutes. In addition, we further explored whether the inhibitor or agonist of SIRT1 could influence of PEPCK1 and found SIRT1 showed a negative affect on PEPCK1. Thus, SIRT2 inhibition, not SIRT1, might serve as a dominant and more efficient way to regulate gluconeogenesis. We propose that sirtinol plays a critical role in coordinating the level of PEPCK1 by SIRT2 inhibition. Consistent with our previous studies, this data demonstrate that SIRT2 is the major enzyme responsible for PEPCK1 acetylation and is the downstream target of sirtinol. Therefore, PEPCK1 stability is controlled by a regulatory network, including the balance between acetylation and deacetylation in response to sirtinol. Employing mass spectrometry experiments and catalytic-mutated phenotypic SIRT2, we found sirtinol to be mainly responsible for the post-translational modification control of PEPCK1, and SIRT2 is the direct target of sirtinol in regulating PEPCK1 degradation. PEPCK1, which reverses the reaction catalyzed by pyruvate kinase in glycolysis, is the critical enzyme for gluconeogenesis and is linked with hepatic glucose output 5 . When sirtinol is present, the acetylated PEPCK1 is ubiquitinylated and subsequently degraded by proteasomes. Therefore, gluconeogenesis is suppressed. As for the acetylation sites, mutation of three lysines to glutamines (an acetylation mimetic) should increase PEPCK1 ubiquitination but it decreased it to the same levels as mutation of the same residues to arginines (a deacetylation mimetic). We believed these lysines residues were specific signals for degradation, modifications of other amino acids could not be recognized by ubiquitin. Our data shows that glucose output and PEPCK1 protein levels, both of which are regulated by sirtinol, were closely correlated in vitro. More importantly, these cells demonstrated tolerance to sirtinol even under as much as 200 μM of sirtinol treatment, which implied that the drug can be used in vivo. Indeed, pyruvate tolerance tests demonstrate that mice can tolerate sirtinol, and sirtinol inhibits the conversion of pyruvate to glucose in vivo.
In the present study, we provide insight into the regulation of sirtinol on cellular gluconeogenesis. Sirtinol decreased liver PEPCK1 protein level and prevented pyruvate-induced blood glucose from increasing. Based on our results, rate-limiting enzyme PEPCK1 is the downstream target of sirtinol, and the inhibition of SIRT2 activity plays an important role in cell gluconeogenesis. Thus, SIRT2 may be a novel molecular target for diabetes therapy, and may shed light on the underlying diabetes treatment mechanism of sirtinol.  penicillin (Invitrogen) (100 U/ml), streptomycin (Invitrogen) (100 U/ml), and 500 nM dexamethasone (dex; Sangon Biotech, Shanghai, China) at a density of 5 × 10 5 cells/well on 6-well plates or 5 × 10 6 cells/100-mm cell culture plate. After attachment (2 hours), the medium was removed and fresh medium was added for 16 hours, followed by drug treatment as described below.

Ethics
Western blot. Standard procedures were followed for western blot, except for the detection of acetylation, which used 50 mM Tris (pH 7.5) with 10% (v/v) Tween 20 and 1% peptone (AMRESCO, Solon, OH, USA) as a blocking buffer. Primary and secondary antibodies were diluted in 50 mM Tris (pH 7.5) with 0.1% peptone. Signals were probed using the chemiluminescence ECL plus reagent (Thermo, Grand Island, NY, USA) and detected using a Typhoon FLA9500 scanner (GE, Fairfield, CT, USA).
Animal Experiments. Mice were purchased from the Department of Laboratory Animal Science, Fudan University. Mice were treated with sirtinol at a dose of 10 mg/kg bodyweight in 100 ml volume intraperitoneal injection twice a week for three weeks. At 1 hour following tail vein injection, blood was collected from the tail vein for glucose detection (Roche). Tissues were collected rapidly from anaesthetized mice and frozen in liquid nitrogen for further analysis.
Statistics. Data was expressed as means ± standard error of the mean (SE). The data was analyzed through one-way ANOVAs followed by post hoc Duncan tests (SPSS 17.0). P < 0.05 was considered significant.

Figure 6.
Sirtinol destabilizes PEPCK1 and inhibits gluconeogenesis in mice. (A) sirt2 +/+ mice (n = 6) were starved overnight, then blood glucose were measured at different time points as indicated. Two groups of mice were injected with saline and sirtinol (10 mg/kg body weight) respectively at the begining. Then pyruvate (2 g/ kg body weight) were injected as indicated. (B) sirt2 +/+ mice were sacrificed after pyruvate tolerance tests, liver tissue were harvested and lysated, then visualized by Western blotting. (C) sirt2 −/− mice (n = 6) were starved for overnight, then blood glucose were measured at different time points as indicated. Two groups of mice were injected with saline and sirtinol (10 mg/kg body weight) respectively at the begining. Then pyruvate (2 g/ kg body weight) were injected as indicated. (D) sirt2 −/− mice were sacrificed after pyruvate tolerance tests, liver tissue were harvested and lysated, then visualized by Western blotting. The data are represented as the means ± SEM. *P < 0.05; **P < 0.01; NS, not significant.