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• W1991782854 abstract "The family of mammalian Sirtuin proteins comprises seven members homologous to yeast Sir2. Here we show that SIRT2, a cytoplasmic sirtuin, is the most abundant sirtuin in adipocytes. Sirt2 expression is downregulated during preadipocyte differentiation in 3T3-L1 cells. Overexpression of SIRT2 inhibits differentiation, whereas reducing SIRT2 expression promotes adipogenesis. Both effects are accompanied by corresponding changes in the expression of PPARγ, C/EBPα, and genes marking terminal adipocyte differentiation, including Glut4, aP2, and fatty acid synthase. The mechanism underlying the effects of reduced SIRT2 in 3T3-L1 adipocytes includes increased acetylation of FOXO1, with direct interaction between SIRT2 and FOXO1. This interaction enhances insulin-stimulated phosphorylation of FOXO1, which in turn regulates FOXO1 nuclear and cytosolic localization. Thus, Sirt2 acts as an important regulator of adipocyte differentiation through modulation of FOXO1 acetylation/phosphorylation and activity and may play a role in controlling adipose tissue mass and function. The family of mammalian Sirtuin proteins comprises seven members homologous to yeast Sir2. Here we show that SIRT2, a cytoplasmic sirtuin, is the most abundant sirtuin in adipocytes. Sirt2 expression is downregulated during preadipocyte differentiation in 3T3-L1 cells. Overexpression of SIRT2 inhibits differentiation, whereas reducing SIRT2 expression promotes adipogenesis. Both effects are accompanied by corresponding changes in the expression of PPARγ, C/EBPα, and genes marking terminal adipocyte differentiation, including Glut4, aP2, and fatty acid synthase. The mechanism underlying the effects of reduced SIRT2 in 3T3-L1 adipocytes includes increased acetylation of FOXO1, with direct interaction between SIRT2 and FOXO1. This interaction enhances insulin-stimulated phosphorylation of FOXO1, which in turn regulates FOXO1 nuclear and cytosolic localization. Thus, Sirt2 acts as an important regulator of adipocyte differentiation through modulation of FOXO1 acetylation/phosphorylation and activity and may play a role in controlling adipose tissue mass and function. The SIR2 (silent information regulator 2) proteins belong to the family of class III NAD-dependent deacetylases that catalyze a reaction in which NAD and an acetylated substrate are converted into a deacetylated protein, nicotinamide, and a novel metabolite O-acetyl ADP-ribose (Tanner et al., 2000Tanner K.G. Landry J. Sternglanz R. Denu J.M. Silent information regulator 2 family of NAD- dependent histone/protein deacetylases generates a unique product, 1-O-acetyl-ADP-ribose.Proc. Natl. Acad. Sci. USA. 2000; 97: 14178-14182Crossref PubMed Scopus (475) Google Scholar). The founding member of the family, sir2, was originally discovered in yeast as a factor that silences the mating-type locus (Imai et al., 2000Imai S. Armstrong C.M. Kaeberlein M. Guarente L. Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase.Nature. 2000; 403: 795-800Crossref PubMed Scopus (2565) Google Scholar, Tanny et al., 1999Tanny J.C. Dowd G.J. Huang J. Hilz H. Moazed D. An enzymatic activity in the yeast Sir2 protein that is essential for gene silencing.Cell. 1999; 99: 735-745Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar). sir2 is also involved in telomere regulation, maintenance of genomic integrity, and lifespan extension in yeast, and similar effects have been shown for its ortholog in C. elegans (Imai et al., 2000Imai S. Armstrong C.M. Kaeberlein M. Guarente L. Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase.Nature. 2000; 403: 795-800Crossref PubMed Scopus (2565) Google Scholar, Wang and Tissenbaum, 2006Wang Y. Tissenbaum H.A. Overlapping and distinct functions for a Caenorhabditis elegans SIR2 and DAF-16/FOXO.Mech. Ageing Dev. 2006; 127: 48-56Crossref PubMed Scopus (210) Google Scholar). In mammals, the homologs of sir2 have been named sirtuins (sirt), with seven members in a family termed sirt1 through sirt7. They share a conserved central deacetylase domain but have different N- and C termini and display distinct subcellular localization, suggesting different biological functions (North and Verdin, 2004North B.J. Verdin E. Sirtuins: Sir2-related NAD-dependent protein deacetylases.Genome Biol. 2004; 5: 224Crossref PubMed Scopus (385) Google Scholar). Mammalian sirt1 is most homologous to yeast sir2 and is found predominantly in the nucleus, consistent with its roles in formation of heterochromatin and gene silencing by histone deacetylation. In mammalian cells, instead of genome silencing, sirt1 often promotes gene transcription by deacetylating specific transcription factors, corepressors, and coactivators, including p53, PGC-1α, NF-kB, MyoD, and members of the FOXO family (Daitoku et al., 2004Daitoku H. Hatta M. Matsuzaki H. Aratani S. Ohshima T. Miyagishi M. Nakajima T. Fukamizu A. Silent information regulator 2 potentiates Foxo1-mediated transcription through its deacetylase activity.Proc. Natl. Acad. Sci. USA. 2004; 101: 10042-10047Crossref PubMed Scopus (478) Google Scholar, Fulco et al., 2003Fulco M. Schiltz R.L. Iezzi S. King M.T. Zhao P. Kashiwaya Y. Hoffman E. Veech R.L. Sartorelli V. Sir2 regulates skeletal muscle differentiation as a potential sensor of the redox state.Mol. Cell. 2003; 12: 51-62Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar, Luo et al., 2001Luo J. Nikolaev A.Y. Imai S. Chen D. Su F. Shiloh A. Guarente L. Gu W. Negative control of p53 by Sir2alpha promotes cell survival under stress.Cell. 2001; 107: 137-148Abstract Full Text Full Text PDF PubMed Scopus (1790) Google Scholar, Nemoto et al., 2005Nemoto S. Fergusson M.M. Finkel T. SIRT1 functionally interacts with the metabolic regulator and transcriptional coactivator PGC-1{alpha}.J. Biol. Chem. 2005; 280: 16456-16460Crossref PubMed Scopus (769) Google Scholar, Yeung et al., 2004Yeung F. Hoberg J.E. Ramsey C.S. Keller M.D. Jones D.R. Frye R.A. Mayo M.W. Modulation of NF-kappaB-dependent transcription and cell survival by the SIRT1 deacetylase.EMBO J. 2004; 23: 2369-2380Crossref PubMed Scopus (1939) Google Scholar). In adipocytes, sirt1 acts as an inhibitor of adipogenesis by interacting with PPARγ corepressor NcoR and SMART, thereby repressing PPARγ activity (Picard et al., 2004Picard F. Kurtev M. Chung N. Topark-Ngarm A. Senawong T. Machado De Oliveira R. Leid M. McBurney M.W. Guarente L. Sirt1 promotes fat mobilization in white adipocytes by repressing PPAR-gamma.Nature. 2004; 429: 771-776Crossref PubMed Scopus (1554) Google Scholar). Much less is known about the function of the other sirtuins. In contrast to SIRT1, mammalian SIRT2 is localized mainly in the cytoplasm. Studies in mammalian cells have suggested that sirt2 may play a role in cell cycle regulation and be involved in cytoskeleton organization by targeting the cytoskeletal protein tubulin (North et al., 2003North B.J. Marshall B.L. Borra M.T. Denu J.M. Verdin E. The human Sir2 ortholog, SIRT2, is an NAD+-dependent tubulin deacetylase.Mol. Cell. 2003; 11: 437-444Abstract Full Text Full Text PDF PubMed Scopus (1123) Google Scholar). The yeast ortholog of sirt2, hst2, has been shown to extend lifespan by a mechanism independent of sir2/hst1 (Lamming et al., 2005Lamming D.W. Latorre-Esteves M. Medvedik O. Wong S.N. Tsang F.A. Wang C. Lin S.J. Sinclair D.A. HST2 mediates SIR2-independent life-span extension by calorie restriction.Science. 2005; 309: 1861-1864Crossref PubMed Scopus (179) Google Scholar). Functional studies showed that sirt3 deacetylates acetyl-CoA synthase 2 (ACS2) and regulates its activity (Hallows et al., 2006Hallows W.C. Lee S. Denu J.M. Sirtuins deacetylate and activate mammalian acetyl-CoA synthetases.Proc. Natl. Acad. Sci. USA. 2006; 103: 10230-10235Crossref PubMed Scopus (606) Google Scholar, Schwer et al., 2006Schwer B. Bunkenborg J. Verdin R.O. Andersen J.S. Verdin E. Reversible Lys acetylation controls the activity of the mitochondrial enzyme acetyl-CoA synthetase 2.Proc. Natl. Acad. Sci. USA. 2006; 103: 10224-10229Crossref PubMed Scopus (524) Google Scholar). The sirt3 also appears to be involved in longevity (Rose et al., 2003Rose G. Dato S. Altomare K. Bellizzi D. Garasto S. Greco V. Passarino G. Feraco E. Mari V. Barbi C. BonaFe M. Franceschi C. Tan Q. Boiko S. Yashin A.I. De Benedictis G. Variability of the SIRT3 gene, human silent information regulator Sir2 homologue, and survivorship in the elderly.Exp. Gerontol. 2003; 38: 1065-1070Crossref PubMed Scopus (225) Google Scholar). The functions of sirt4–7 are less clear; recent reports have shown that sirt6 may also be involved in aging in mice, while sirt7 appears to regulate DNA pol I transcription (Ford et al., 2006Ford E. Voit R. Liszt G. Magin C. Grummt I. Guarente L. Mammalian Sir2 homolog SIRT7 is an activator of RNA polymerase I transcription.Genes Dev. 2006; 20: 1075-1080Crossref PubMed Scopus (431) Google Scholar, Mostoslavsky et al., 2006Mostoslavsky R. Chua K.F. Lombard D.B. Pang W.W. Fischer M.R. Gellon L. Liu P. Mostoslavsky G. Franco S. Murphy M.M. et al.Genomic instability and aging-like phenotype in the absence of mammalian SIRT6.Cell. 2006; 124: 315-329Abstract Full Text Full Text PDF PubMed Scopus (1085) Google Scholar). Mammalian forkhead transcription factors of class O (FoxO) include foxO1, foxO3a, and foxO4 and have been shown to be involved with wide range of cellular processes, such as DNA repair, cell cycle control, stress resistance, apoptosis, and metabolism (Barthel et al., 2005Barthel A. Schmoll D. Unterman T.G. FoxO proteins in insulin action and metabolism.Trends Endocrinol. Metab. 2005; 16: 183-189Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar, Furukawa-Hibi et al., 2005Furukawa-Hibi Y. Kobayashi Y. Chen C. Motoyama N. FOXO transcription factors in cell-cycle regulation and the response to oxidative stress.Antioxid. Redox Signal. 2005; 7: 752-760Crossref PubMed Scopus (163) Google Scholar). Among all FoxO members, foxo1 appears to have an important role in adipocyte differentiation, acting as an inhibitor of adipogenesis at an early phase of the differentiation process (Nakae et al., 2003Nakae J. Kitamura T. Kitamura Y. Biggs W.H. Arden K.C. Accili D. The forkhead transcription factor Foxo1 regulates adipocyte differentiation.Dev. Cell. 2003; 4: 119-129Abstract Full Text Full Text PDF PubMed Scopus (564) Google Scholar). In this context, the enzyme phosphatidylinositol 3-kinase (PI-kinase), which is stimulated by insulin and certain cytokines and growth factors, can negatively regulate FoxOs (Zhang et al., 2002Zhang X. Gan L. Pan H. Guo S. He X. Olson S.T. Mesecar A. Adam S. Unterman T.G. Phosphorylation of Ser 256 suppresses transactivation by FKHR (FOXO1) by multiple mechanisms. Direct and indirect effects on nuclear/cytoplasmic shuttling and DNA binding.J. Biol. Chem. 2002; 277: 45276-45284Crossref PubMed Scopus (241) Google Scholar). This inhibitory effect of insulin is mainly mediated by Akt/PKB phosphorylation of FOXO, which promotes the trafficking of FOXO from the nucleus to the cytosol. The transcriptional activity of FOXO proteins can also be regulated by acetylation and deacetylation. FOXO1 can be acetylated by CBP acetyl-transferase, and SIRT1 has been shown to deacetylate FOXO1 and regulate its activity, especially under conditions of stress (Matsuzaki et al., 2005Matsuzaki H. Daitoku H. Hatta M. Aoyama H. Yoshimochi K. Fukamizu A. Acetylation of Foxo1 alters its DNA-binding ability and sensitivity to phosphorylation.Proc. Natl. Acad. Sci. USA. 2005; 102: 11278-11283Crossref PubMed Scopus (339) Google Scholar, van der Heide and Smidt, 2005van der Heide L.P. Smidt M.P. Regulation of FoxO activity by CBP/p300-mediated acetylation.Trends Biochem. Sci. 2005; 30: 81-86Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Recently, it has been shown that the extent of deacetylation of FOXO1 can affect its phosphorylation and DNA binding activity to target gene promoters (Matsuzaki et al., 2005Matsuzaki H. Daitoku H. Hatta M. Aoyama H. Yoshimochi K. Fukamizu A. Acetylation of Foxo1 alters its DNA-binding ability and sensitivity to phosphorylation.Proc. Natl. Acad. Sci. USA. 2005; 102: 11278-11283Crossref PubMed Scopus (339) Google Scholar). In the present study, we demonstrate that sirt2 mRNA is more abundant than other sirtuins in adipocytes in vivo and in culture and that sirt2 exerts an inhibitory effect on adipocyte differentiation. This is mediated by regulating FOXO1 deacetylation, leading to changes in FOXO1 phosphorylation, increased nuclear localization, and inhibition of adipogenesis. Thus, activators of sirt2 could provide novel therapeutics of obesity and its complications. Various isoforms of mammalian Sirt proteins are expressed in adipose tissue and 3T3-L1 preadipocytes, with varying expression patterns during differentiation. Affymetrix microarrays that were performed on isolated adipocytes indicated that sirt1, sirt2, and sirt3 were all expressed in adipocytes and that the level of sirt2 was much higher than that of sirt1 or sirt3 (Figure 1A, top panel). Using quantitative real-time PCR with cDNA standard curves for each isoform as described in Experimental Procedures, the molar amounts of different Sirt transcripts per microgram of total RNA were obtained. As shown in Figure 1A (lower panel), the molar amount of sirt2 mRNA per microgram total RNA in 3T3-L1 preadipocytes was 4–5× that of sirt1 and 6–7× that of sirt3. Similar studies including other sirt members confirmed that sirt2 transcripts are more abundant than others (Figure S1). During the first 2 days of differentiation (i.e., the induction phase), levels of both sirt1 and sirt2 mRNA decreased by 60%–70% and then remained stable for the remainder of the time course of differentiation (Figure 1B, top and middle panels). Sirt3 mRNA on the other hand, started at a low level compared to both sirt2 and sirt1, then increased by 3- to 4-fold during adipocyte differentiation (Figure 1B, bottom panel). To investigate the potential role of sirt2 in preadipocytes, we used retroviruses to generate 3T3-L1 stable cell lines carrying either shRNAs targeting endogenous sirt2 or GFP as a control. Since there was no good antibody available for mouse SIRT2 at the time these experiments were preformed, we used real-time PCR to assess sirt2 mRNA levels. This revealed that, compared with shGFP cells, cells stably expressing the two shsirt2 retroviruses exhibited an 80%–90% knockdown of sirt2 mRNA, with no significant change in the level of sirt1 or sirt3 mRNAs (Figure 1C). Furthermore, when the same two retroviral constructs were transiently cotransfected into HEK293 with a CMV-driven SIRT2-FLAG construct, there was a parallel 80%–90% reduction of the tagged SIRT2 protein when compared with cells cotransfected with shGFP (Figure 1D). Thus, the expression of both shsirt2 constructs produced major reductions of sirt2 at the RNA and protein levels, and this reduction was specific to the sirt2 isoform. Later commercial availability of SIRT2 antibody allowed us to detect a similar decrease of endogenous SIRT2 protein in shsirt2 cells (Figure 1D). Preadipocytes stably transfected with either shsirt2 or shGFP were then subjected to adipogenic differentiation protocol, and samples from different time points were collected for either RNA or protein analysis. Oil Red O staining during the time course of differentiation confirmed the increased rate and extent of differentiation, with increased staining of cells by day 4 indicating more rapid accumulation of lipid in sirt2 knockdown cells (Figure 2A). As noted above, in control shGFP-expressing cells sirt2 mRNA expression decreased during the time course of differentiation, while in the shsirt2 -expressing cells, endogenous sirt2 mRNA as assessed by real-time PCR was reduced by 75%–80%, and this persisted throughout the time course of adipocyte differentiation (Figure 2B). As expected, sirt2 knockdown had no significant effect on levels of Sirt1 mRNA or on the change in Sirt1 that occurred during differentiation, consistent with the specificity of sirt2 knockdown (Figure 2B). By contrast, in the sirt2 knockdown cells, two transcription factors central to adipogenic differentiation, C/EBPα and PPARγ, both demonstrated significantly accelerated and exaggerated increase in mRNA expression. Thus, C/EBPα mRNA level was elevated more than 3-fold in shsirt2 cells on day 2 after induction compared with control cells (p = 0.001), and this difference remained throughout the time course of differentiation (Figure 2B). PPARγ mRNA levels in shsirt2 cells were also 2- to 3-fold higher than in shGFP cells after induction and throughout the time course, with the greatest increase on day 2 (Figure 2B). Corresponding to elevated early adipogenic transcription factor expression, mRNA levels of various late adipocyte differentiation markers that are downstream C/EBPα and PPARγ (Lane et al., 1999Lane M.D. Tang Q.Q. Jiang M.S. Role of the CCAAT enhancer binding proteins (C/EBPs) in adipocyte differentiation.Biochem. Biophys. Res. Commun. 1999; 266: 677-683Crossref PubMed Scopus (232) Google Scholar, Qi et al., 2000Qi C. Zhu Y. Reddy J.K. Peroxisome proliferator-activated receptors, coactivators, and downstream targets.Cell Biochem. Biophys. 2000; 32: 187-204Crossref PubMed Scopus (188) Google Scholar) were also significantly enhanced in shsirt2 cells during the time course of differentiation. For example, on day 2 after induction, shsirt2 cells had ∼3-fold higher levels of aP2 mRNA (p = 0.002) and ∼2-fold higher levels of fatty acid synthase (FAS; p = 0.0014) and Glut 4 mRNA (p = 0.0055) when compared to control cells (Figure 2B). On the other hand, expression of foxo1 showed no significant change at mRNA level at any time point during differentiation (Figure 2B). Western blot analysis of proteins confirmed these effects of sirt2 knockdown on expression of adipocyte differentiation markers (Figure 2C). On day 2 after induction, there was a 2-fold increase in C/EBPβ and a 5-fold increase in C/EBPα protein in shsirt2 cells compared to control, and this increase in C/EBPα persisted through differentiation, even as levels in the control cells increased. A similar pattern of increased protein expression was observed for PPARγ protein in shsirt2 cells. The increase was even more marked for the late adipocyte differentiation marker, FAS, which was 4-fold elevated at the protein level in shsirt2 cells on day 2 compared to controls, although this difference diminished on day 8 as the cells became mature and FAS expression increased in the control cells (Figure 2C). Endogenous SIRT2 protein expression was consistent with its mRNA expression during differentiation in shGFP cells, while endogenous SIRT2 protein was knocked down in shsirt2 cells. Opposite effects were observed in 3T3-L1 cells overexpressing sirt2. Overexpression of SIRT2-FLAG in 3T3-L1 cells inhibited adipocyte differentiation and lipid accumulation compared with empty vector control cells (Figure 3A). Western blot analysis of adipocyte markers, such as PPARγ and FAS, also revealed decreased levels in sirt2-overexpressing cell line (Figure 3A). As insulin signaling pathway is one of the major pathways that controls adipogenesis and adipocyte differentiation, we tested if the effect of sirt2 on 3T3-L1 differentiation was due to altered insulin signaling. Acute (10 min) insulin stimulation of both control and sirt2-overexpressing cell lines produced equal phosphorylation responses for Akt, p42/p44 MAP kinase, and p38 MAP kinase (Figure 3B). Thus, overexpression of sirt2 in 3T3-L1 cells inhibits the normal adipogenic process, and this effect occurs without a change in upstream insulin signaling. Conversely, reducing sirt2 expression enhances the program of adipogenic gene expressions at the mRNA and protein levels, and this is associated with enhanced lipid accumulation. The subcellular localization of SIRT2-FLAG overexpression is similar to previous reports that SIRT2 is mainly a cytoplasmic protein (Figure S2). Foxo1, a known inhibitor of adipogenesis, has been previously shown to undergo regulated acetylation and deacetylation (Matsuzaki et al., 2005Matsuzaki H. Daitoku H. Hatta M. Aoyama H. Yoshimochi K. Fukamizu A. Acetylation of Foxo1 alters its DNA-binding ability and sensitivity to phosphorylation.Proc. Natl. Acad. Sci. USA. 2005; 102: 11278-11283Crossref PubMed Scopus (339) Google Scholar, Perrot and Rechler, 2005Perrot V. Rechler M.M. The coactivator p300 directly acetylates the forkhead transcription factor Foxo1 and stimulates Foxo1-induced transcription.Mol. Endocrinol. 2005; 19: 2283-2298Crossref PubMed Scopus (92) Google Scholar, Daitoku et al., 2004Daitoku H. Hatta M. Matsuzaki H. Aratani S. Ohshima T. Miyagishi M. Nakajima T. Fukamizu A. Silent information regulator 2 potentiates Foxo1-mediated transcription through its deacetylase activity.Proc. Natl. Acad. Sci. USA. 2004; 101: 10042-10047Crossref PubMed Scopus (478) Google Scholar). Since there was no change in foxo1 expression at the mRNA level, we explored whether FOXO1 protein expression or acetylation might be changed. Immunoprecipitation using anti-acetyl-Lys antibody followed by blotting with anti-FOXO1 antibody revealed that in control shGFP cells, most of the FOXO1 protein was in a deacetylated state (i.e., FOXO1 could not be detected in precipitated total acetylated protein). By contrast, in the Sirt2 knockdown cells, FOXO1 acetylation was markedly increased, and the anti-FOXO1 antibody easily detected the presence of FOXO1 protein in the precipitated lysate (Figure 4). This effect was specific because western blot analysis with anti-FoxO3a antibody did not detect any increased protein acetylation (data not shown). To verify the increased FOXO1 acetylation in Sirt2 knockdown cells, the IP of exogenous FOXO1-FLAG protein was done with both shGFP and shsirt2 cell lysates; the results indicate that while a similar amount of FOXO1-FLAG protein is precipitated from either cell line, there is increased acetylation on FOXO-FLAG precipitated from shsirt2 cell lysate (Figure S3). These effects on FOXO1 acetylation occurred with no change in the total level of FOXO1 protein in sirt2 knockdown cells and no change in the level of Sirt1 protein (Figure 4), another member of the Sirt family, which is able to deacetylate FoxOs. The increased acetylation on FOXO1 in sirt2 knockdown cells indicates that foxo1 can serve as a potential target for SIRT2 deacetylase activity. To investigate if SIRT2 interacts with FOXO1 directly, we performed immunoprecipitation of total cell lysates of cells overexpressing SIRT2-FLAG versus control cells infected with the empty pBabe retrovirus using a monoclonal anti-FLAG antibody conjugated to agarose. The immunoprecipitates were then immunoblotted with anti-FOXO1 antibody. In the cells expressing the SIRT2-FLAG construct, the anti-FLAG antibody coprecipitated significantly more FOXO1 protein than in control cells (Figure 5A), indicating that SIRT2 is present in a complex with FOXO1 protein. Western blot analysis of the same cell lysates with anti-FOXO1 antibody showed that this occurred with no difference in total FOXO1 protein content between SIRT2-FLAG and control cell lines (Figure 5A). The unchanged FOXO1 protein levels in preadipocytes of both sirt2 knockdown and -overexpressing cells is consistent with the real-time PCR data indicating that foxo1 mRNA expression was not altered in sirt2 knockdown cells during 3T3-L1 differentiation (Figure 2B). In lysates from cells overexpressing recombinant SIRT2-HA and FOXO1-FLAG, SIRT2-HA can coimmunoprecipitate FOXO1-FLAG in vitro (Figure 5B), confirming the interaction between SIRT2 and FOXO1 protein. To determine if the increased acetylation of FOXO1 could alter its ability to undergo phosphorylation, we treated serum-deprived shsirt2 and shGFP preadipocytes with insulin at different concentrations and immunoblotted cell extracts with an antibody that detects phosphorylation of FOXO1 Ser-253, the major site of FOXO1 phosphorylation by Akt/PKB (van der Heide et al., 2004van der Heide L.P. Hoekman M.F. Smidt M.P. The ins and outs of FoxO shuttling: mechanisms of FoxO translocation and transcriptional regulation.Biochem. J. 2004; 380: 297-309Crossref PubMed Scopus (525) Google Scholar). Consistent with the data above, insulin stimulated Akt/PKB phosphorylation to the same level in the sirt2 knockdown and control cell lines. On the other hand, phosphorylation of FOXO1 on Ser-253 was increased 2-fold in the sirt2 knockdown cell line (Figure 5C). Since phosphorylation is known to affect nuclear translocation, nuclear and cytosolic extracts from shGFP and shsirt2 cells were prepared and subjected to immunoblot analysis with anti-FOXO1 antibody. This revealed a 2- to 3-fold increase in the level of cytosolic FOXO1 protein in Sirt2 knockdown cells. Also, it was clear that the cytosolic FOXO1 band migrated in a slightly retarded position on the gel in the shsirt2 cells, consistent with increased FOXO1 phosphorylation, whereas the nuclear FOXO1 protein migrated at a lower position on the gels due to its unphosphorylated state. Furthermore, nuclear FOXO1 was decreased in amount (Figure 5D). Due to high background of FOXO1 antibody, we generated 3T3 L1 cell lines overexpressing FLAG-tagged FOXO1 along with either shGFP or shsirt2 stable constructs, then used anti-FLAG to detect the subcellular localization of recombinant FOXO1. FOXO1-FLAG was largely excluded from the nucleus of cells overexpressing shsirt2, while cells overexpressing shGFP showed a more diffusive pattern of FOXO1-FLAG localization (Figure 5D). Immunoblot of total protein lysates with anti-FOXO1 antibody revealed no difference in total FOXO1 protein between the two lines (Figure 5C). To validate the effect of foxo1 on adipocyte differentiation, an shRNA-mediated endogenous foxo1 knockdown experiment was done, and the effects of foxo1 knockdown on adipogenesis were accessed. The results showed that reducing endogenous foxo1 expression efficiently promoted adipocyte differentiation with increased Oil Red O staining accompanied by increased adipocyte marker expression, such as PPARγ and C/EBPα (Figure S4). To further analyze the possible role of FOXO1 acetylation in the regulation of FOXO1 phosphorylation, we used 3T3-L1 cell lines overexpressing either wild-type foxo1 or two foxo1 mutants that mimic different acetylation states of the protein. In the KQ mutant, the three Lys residues surrounding Ser-253 that are known to be sites of acetylation were replaced by glutamine residues. In the KR mutant, these Lys were replaced by Arg residues. All three overexpression constructs were generated with an N-terminal FLAG tag to allow quantitation of the protein. Immunoblotting of lysates from confluent cells overexpressing either foxo1 wild-type or the KQ and KR mutants with anti-FLAG monoclonal antibody revealed that all three proteins were equally overexpressed (Figure 6A). Quantitative PCR indicated a 5-fold increase in total foxo1 mRNA in each line as compared to endogenous foxo1 levels (Figure S5). Quantitative PCR using primers targeting the untranslated region of endogenous foxo1 mRNA demonstrated that the endogenous foxo1 expression level was not affected by expression of the exogenous protein (data not shown). The cell lines overexpressing wild-type and mutant foxo1 were subjected to the standard adipogenic differentiation protocol and stained with Oil Red O. Cells overexpressing wild-type foxo1 showed much less Oil Red O staining, consistent with a significantly decreased level of differentiation, than cells infected with the empty vector. This finding is consistent with the known ability of foxo1 to suppress adipogenesis. The cells overexpressing the KQ mutant of foxo1, which mimics the acetylated state, exhibited enhanced differentiation compared with cells overexpressing wild-type foxo1. In contrast, cells overexpressing the KR mutant, which mimics the deacetylated protein, showed decreased differentiation compared with cells overexpressing wild-type foxo1 (Figure 6A). These differences in lipid accumulation correlated well with expression of different adipocyte differentiation markers such as aP2, PPARγ, and C/EBPα by quantitative PCR (Figure 6A). Assessment of FOXO1 Ser-253 phosphorylation after insulin stimulation in these cell lines revealed increased phosphorylation of the KQ mutant in the basal state, as well as a substantially higher level of phosphorylation following insulin stimulation when compared with cells overexpressing wild-type protein. By contrast, cells expressing the KR mutant of foxo1 showed decreased Ser-253 phosphorylation in the insulin-stimulated condition (Figure 6B). Thus, the FOXO1 acetylation mimic had increased Ser-253 phosphorylation, whereas the deacetylated FOXO1 mimic had decreased Ser-253 phosphorylation. The FLAG western blot shows that the total recombinant FOXO1 protein expression is not altered under above conditions. These changes on FOXO1 phosphorylation occurred with no change in the level of phosphorylated/activated Akt and phosphorylated GSK3β (Figure 6B). The subcellular localization of FOXO1 mutants detected by immunocytochemistry using anti-FLAG-FITC was consistent with the observed differences in localization by subcellular fractionation and FOXO1 phosphorylation. Both foxo1 wild-type and KR overexpression had a diffused distribution within the cell, but KQ mutant had a nuclear exclusion pattern, where the KQ was more phosphorylated and localized in the cytoplasm (Figure 6B). The sirtuins represent a complex family of proteins that show homology to the yeast class III NAD-dependent protein/histone deacetylase sir2. Sirt1, the be" @default.
• W1991782854 created "2016-06-24" @default.
• W1991782854 creator A5008583934 @default.
• W1991782854 creator A5051737440 @default.
• W1991782854 creator A5079571269 @default.
• W1991782854 date "2007-08-01" @default.
• W1991782854 modified "2023-10-12" @default.
• W1991782854 title "SIRT2 Regulates Adipocyte Differentiation through FoxO1 Acetylation/Deacetylation" @default.
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