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• W2598459868 abstract "Article31 March 2017free access Source DataTransparent process The phosphorylation status of T522 modulates tissue-specific functions of SIRT1 in energy metabolism in mice Jing Lu Signal Transduction Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA Department of Pathology, Wake Forest School of Medicine, Winston-Salem, NC, USA Search for more papers by this author Qing Xu Signal Transduction Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA Search for more papers by this author Ming Ji Signal Transduction Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA Search for more papers by this author Xiumei Guo Signal Transduction Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA Search for more papers by this author Xiaojiang Xu Integrative Bioinformatics, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA Search for more papers by this author David C Fargo Integrative Bioinformatics, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA Search for more papers by this author Xiaoling Li Corresponding Author [email protected] orcid.org/0000-0001-5920-7784 Signal Transduction Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA Search for more papers by this author Jing Lu Signal Transduction Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA Department of Pathology, Wake Forest School of Medicine, Winston-Salem, NC, USA Search for more papers by this author Qing Xu Signal Transduction Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA Search for more papers by this author Ming Ji Signal Transduction Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA Search for more papers by this author Xiumei Guo Signal Transduction Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA Search for more papers by this author Xiaojiang Xu Integrative Bioinformatics, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA Search for more papers by this author David C Fargo Integrative Bioinformatics, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA Search for more papers by this author Xiaoling Li Corresponding Author [email protected] orcid.org/0000-0001-5920-7784 Signal Transduction Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA Search for more papers by this author Author Information Jing Lu1,2,‡, Qing Xu1,‡, Ming Ji1,‡, Xiumei Guo1,†,‡, Xiaojiang Xu3, David C Fargo3 and Xiaoling Li *,1 1Signal Transduction Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA 2Department of Pathology, Wake Forest School of Medicine, Winston-Salem, NC, USA 3Integrative Bioinformatics, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA †Present address: Guangzhou DiagCor Clinical Laboratory Co., Ltd, Guangzhou, Guangdong, China ‡These authors contributed equally to this work *Corresponding author. Tel: +1 919 541 9817; E-mail: [email protected] EMBO Rep (2017)18:841-857https://doi.org/10.15252/embr.201643803 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract SIRT1, the most conserved mammalian NAD+-dependent protein deacetylase, is an important metabolic regulator. However, the mechanisms by which SIRT1 is regulated in vivo remain unclear. Here, we report that phosphorylation modification of T522 on SIRT1 is crucial for tissue-specific regulation of SIRT1 activity in mice. Dephosphorylation of T522 is critical for repression of its activity during adipogenesis. The phospho-T522 level is reduced during adipogenesis. Knocking-in a constitutive T522 phosphorylation mimic activates the β-catenin/GATA3 pathway, repressing PPARγ signaling, impairing differentiation of white adipocytes, and ameliorating high-fat diet-induced dyslipidemia in mice. In contrast, phosphorylation of T522 is crucial for activation of hepatic SIRT1 in response to over-nutrition. Hepatic SIRT1 is hyperphosphorylated at T522 upon high-fat diet feeding. Knocking-in a SIRT1 mutant defective in T522 phosphorylation disrupts hepatic fatty acid oxidation, resulting in hepatic steatosis after high-fat diet feeding. In addition, the T522 dephosphorylation mimic impairs systemic energy metabolism. Our findings unveil an important link between environmental cues, SIRT1 phosphorylation, and energy homeostasis and demonstrate that the phosphorylation of T522 is a critical element in tissue-specific regulation of SIRT1 activity in vivo. Synopsis The phosphorylation of SIRT1 T522 is a critical element in the tissue-specific regulation of SIRT1 activity in mice. This finding unveils an important link between environmental cues, SIRT1 phosphorylation and energy homeostasis. SIRT1 is dephosphorylated at T522 during normal adipogenesis. Knocking-in a constitutive T522 phosphorylation mimic activates the β-catenin/GATA3 pathway, repressing PPARγ signaling and adipogenesis in mice. Hepatic SIRT1 is hyperphosphorylated at T522 upon high-fat diet feeding. Knocking-in a SIRT1 mutant defective in T522 phosphorylation disrupts hepatic fatty acid oxidation, resulting in hepatic steatosis in mice on high-fat diet. Introduction Metabolic syndrome is defined as a cluster of metabolism-related disorders, such as central obesity, type 2 diabetes, dyslipidemia, and high blood pressure, all of which are considered as major contributors of mortality in industrialized countries 1234. Both genetic factors and environmental influences contribute to the pathogenesis of metabolic syndrome. Among which, a class III histone deacetylase and a mammalian homologue of yeast silent information regulator (Sir2) protein, SIRT1, play a central role in the regulation of transcriptional networks in various critical metabolic processes in multiple tissues. For example, SIRT1 is a key modulator of both glucose and fatty acid metabolism in the liver 56. Knocking-down or deletion of hepatic SIRT1 impairs fatty acid oxidation, thereby increasing the susceptibility of mice to dyslipidemia and hepatic steatosis 789. Conversely, hepatic overexpression of SIRT1 attenuates hepatic steatosis and ER stress and restores glucose homeostasis in mice 10. SIRT1 is also an important regulator of maturation and remodeling of adipose tissues 6. It has been reported that SIRT1 represses a master regulator of adipogenesis in the white adipose tissue (WAT), PPARγ, thereby suppressing the expression of adipose tissue markers, such as a fatty acid binding protein, aP2, and inhibiting fat mobilization in response to fasting 11. Moreover, genetic ablation of SIRT1 in adipose tissues leads to increased adiposity and insulin resistance 12, whereas treatment of mice on a high-fat diet with resveratrol, a polyphenol that activates SIRT1 in cells directly or indirectly 1314151617, protects animals against high-fat induced obesity and metabolic dysfunctions 181920. Therefore, current studies point to the notion that SIRT1 functions as an adaptor that is “beneficial” to cellular and organismal metabolism. Consequently, dysfunction of this sirtuin contributes to the development a number of human metabolic diseases, particularly metabolic syndrome 5. Although the role of SIRT1 in metabolic regulation of a variety of biological processes has been well studied, how the activity of SIRT1 is regulated in vivo in response to different biological/environmental cues remains elusive, and the functional/physiological consequences of disruption of its regulation are still unclear. As a NAD+-dependent protein deacetylase, the activity of SIRT1 is tightly controlled at multiple levels, either by the cellular levels of NAD+, which are hypersensitive to a number of environmental cues including fasting, caloric restriction, exercise, or high-fat diet feeding, and/or at the posttranslational level by small chemicals, protein–protein interactions, or through posttranslational modification 21. In particular, we have previously reported that SIRT1 can be phosphorylated and activated by two anti-apoptotic members of the dual-specificity tyrosine phosphorylation-regulated kinase (DYRK), DYRK1A and DYRK3, in response to acute environmental stresses 22. We further showed that this modification activates its deacetylase activity independently of the cellular NAD+ level through preventing the formation of less-active SIRT1 oligomers/aggregates 23. Our findings suggest that phosphorylation modification of SIRT1 might provide a molecular mechanism that fine-tunes SIRT1 activity in vivo independently of the cellular NAD+ level. To assess the physiological impacts of the phosphorylation of T522 on SIRT1, we generated SIRT1 T522 phosphorylation mimic (threonine to glutamic acid, or T522E mutation, or TE) and dephosphorylation mimic (threonine to alanine, or T522A mutation, or TA) knock-in mouse models. In this report, we show for the first time that the phosphorylation of T522 renders tissue-specific regulation of SIRT1 activity in response to developmental and nutritional signals in vivo. Results Generation of SIRT1 TEKI and TAKI mice To investigate whether phosphorylation of T522 of SIRT1 is an important posttranslational modification that modulates SIRT1 activity in vivo, we used standard gene-targeting technology to generate two different SIRT1 mutation knock-in mouse lines, SIRT1 TAKI (TAKI) and SIRT1 TEKI (TEKI), with T522A (TA) to mimic the dephosphorylated SIRT1 and T522E (TE) to mimic the phosphorylated SIRT1, respectively (Figs 1 and EV1). Both KI strains were born at the expected Mendelian ratio with no gross phenotypes, indicating that the phosphorylation status of T522 does not affect normal animal development and survival. Immunoblotting analysis of total protein lysates from different tissues with anti-SIRT1 antibody revealed that the protein levels of two mutant proteins in these two lines were comparable to those of wild-type (WT) SIRT1 (Fig 1A). Further analyses with the anti-p-SIRT1(T522) antibody confirmed that knocking-in the TA mutant abolished the phosphorylation signals on the endogenous SIRT1 protein (Fig 1B, Lane TAKI). Since our p-SIRT1(T522) antibody only binds weakly to the phospho-mimics 22, knocking-in the TE mutation also yielded a mutant SIRT1 protein undetectable by this antibody (Fig 1B, Lane TEKI). Figure 1. Generation of SIRT1 TEKI and TAKI mice The SIRT1 expression levels in wild type (WT), TEKI, and TAKI mice are comparable. Total protein lysates from indicated tissues were analyzed by immunoblotting with an anti-SIRT1 antibody. Endogenous SIRT1 proteins from indicated tissues from both TEKI and TAKI mice display decreased p-SIRT1(T522) levels. Total protein lysates from indicated tissues were analyzed by immunoblotting with antibodies against SIRT1 and p-SIRT1(T522). Please note that the p-SIRT1(T522) antibody only displays weak affinity to the SIRT1TE protein. SIRT1 TEKI MEFs have an increased deacetylase activity toward p53 in response to genotoxic stress. MEFs isolated from WT, TEKI, and TAKI mice were treated with adriamycin (0.2 μg/ml) for 8 h or with PBS (as control). Total cell lysates were immunoblotted with acetyl-p53 (K382) or p53 antibodies. SIRT1 TEKI MEFs have elevated deacetylase activity to the p65 subunit of NF-κB in response to inflammatory signals. WT, TEKI, and TAKI MEFs were treated with 10 ng/ml of TNFα, or 1 μg/ml of Escherichia coli O111:B4 lipopolysaccharide (LPS) for 30 min with PBS (as control). Total cell lysates were immunoblotted with acetyl-p65 and p65 antibodies. Source data are available online for this figure. Source Data for Figure 1 [embr201643803-sup-0005-SDataFig1.zip] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Generation of SIRT1 TEKI and TAKI mice Schematic representation of knock-in strategies for generating SIRT1 TEKI and SIRT1 TAKI strains. Locations and sequences of genotyping primers are labeled. Representation images of genotyping PCR results. Primers and qPCR results for specific detection of WT, TEKI, and TAKI mRNA (n = 4 mice for each group). Data Information: In (C), data are presented as mean ± SEM. Download figure Download PowerPoint Our previous studies have demonstrated that in cultured cells, the phospho-mimics of SIRT1 T522 are hyperactive toward acetyl-p53 protein upon genotoxic stress, whereas the dephospho-mimics of SIRT1 T522 are partially inactive 2223. To confirm the changes of SIRT1 deacetylase activity in the knock-in mouse lines, we isolated mouse embryonic fibroblasts (MEFs) from WT, TAKI, and TEKI embryos and analyzed cellular SIRT1 activities. As shown in Fig 1C, acetyl-p53 levels in WT MEFs were increased when treated with a DNA intercalating drug, adriamycin (Adria). This increase was blunted in TEKI MEFs (top panels), indicating that the TE mutant protein has an increased deacetylase activity toward acetyl-p53. The knocked-in SIRT1 TA protein, on the other hand, did not appear to have defects in deacetylation of p53 protein in response to adriamycin-induced genotoxic stress (bottom panels), possibly due to stable knock-in induced compensatory effects in regulating p53 acetylation in these cells. However, both TE and TA mutants displayed expected enhanced (TEKI, top panels) and reduced (TAKI, bottom panels) deacetylase activities toward acetyl-p65 when MEFs were challenged with pro-inflammatory stimuli, TNFα or LPS (Fig 1D). Taken together, our observations indicate that when stably knocked-in mice, the SIRT1 TEKI allele exhibits the expected enhanced activity upon all tested environmental stress, while the TAKI allele displays the expected activity in response to specific environmental stimuli. Phosphorylation of SIRT1 at T522 inhibits adipogenesis in vitro SIRT1 is a negative regulator of adipogenesis 6, suggesting that the activity of this sirtuin must be repressed during the process of normal adipogenesis. To test whether phosphorylation of T522 plays a role in regulation of SIRT1 activity in this process, we analyzed the p-SIRT1(T522) levels of endogenous SIRT1 protein during a 7-day in vitro adipogenesis of WT primary MEFs. As shown in Fig 2A, p-SIRT1 levels were gradually reduced while total SIRT1 protein remained constant during in vitro adipogenesis, indicating that SIRT1 phosphorylation (activity) but not SIRT1 expression is negatively correlated with adipogenesis. Moreover, this reduction in p-SIRT1 levels was accompanied with decreased levels of DYRK3 but not DYRK1A (Fig 2A and B), suggesting that reduced expression of DYRK3 might be a reason for the diminished phosphorylation of SIRT1 in this process. Figure 2. The SIRT1 TEKI allele inhibits adipogenesis in vitro The endogenous SIRT1 protein is dephosphorylated at T522 during in vitro differentiation of MEFs into adipocytes. Primary MEFs isolated from WT mice were treated and induced to differentiation into adipocytes in vitro as described in Materials and Methods. The levels of indicated proteins were analyzed by immunoblotting. The expression levels of Dyrk3 but not Dyrk1a are decreased during in vitro adipogenesis (n = 3 independent experiments). The mRNA levels of indicated genes were analyzed by qPCR. SIRT1 TEKI MEFs have reduced in vitro adipogenesis. In vitro differentiated adipocytes from primary MEFs isolated from WT, TAKI, and TEKI mice were stained by Oil Red O. Scale bars, 50 μm. In vitro differentiated adipocytes from primary TEKI MEFs have reduced expression levels of adipocyte markers (n = 3 independent experiments). Data Information: In (B and D), data are presented as mean ± SEM. *P < 0.05 (Mann–Whitney test). Source data are available online for this figure. Source Data for Figure 2 [embr201643803-sup-0006-SDataFig2.pdf] Download figure Download PowerPoint In line with above observations, primary MEFs isolated from dephosphorylation defective TEKI mice accumulated much less fat as determined by Oil Red O staining after 7 days of differentiation compared with WT MEFs, whereas MEFs from constitutively dephosphorylated TAKI mice displayed a comparable ability to be differentiated into adipocytes as WT MEFs (Fig 2C). Consistently, the mRNA levels of PPARγ2, a nutrition-sensitive isoform of PPARγ, aP2, as well as Glut4, an adipose tissue-specific glucose transporter, were significantly reduced in TEKI but not in TAKI cells during differentiation (Fig 2D). Taken together, our finding indicates that dephosphorylation of SIRT1, thereby reduction in SIRT1 activity, is required for normal adipogenesis in vitro. The SIRT1 TEKI allele actives β-catenin/GATA3 signaling, repressing PPARγ and impairing functions of WAT in vitro and in vivo Although SIRT1 has been shown to indirectly repress or directly activate PPARγ in response to different environmental signals in WAT 1124, the reduced induction of PPARγ2 during adipogenesis of TEKI cells (Fig 2D) raises the possibility that the SIRT1 TE mutant protein may blunt the transcription of PPARγ2, thereby repressing adipocyte differentiation. To test this possibility, we analyzed the expression of several genes that have been previously shown to be important in transcriptional regulation of PPARγ, such as β-catenin, GATA2, and GATA3, in WT and TEKI MEFs before and after differentiation into adipocytes. As shown in Fig 3A–C, both mRNA and protein levels of GATA3 but not its close homolog GATA2 nor β-catenin were significantly elevated in TEKI MEFs before and at the early stages of differentiation. Figure 3. The SIRT1 TEKI allele actives β-catenin/GATA3 signaling TEKI MEFs have increased expression levels of GATA3 during in vitro adipogenesis (n = 3 independent experiments). The mRNA levels of indicated genes were analyzed by qPCR. Primary TEKI MEFs have increased mRNA levels of GATA3 but not β-catenin (n = 4 independent experiments). The mRNA levels of indicated genes were analyzed by qPCR. Primary TEKI MEFs have increased protein levels of GATA3 but reduced β-catenin protein. The levels of indicated protein were analyzed by immunoblotting. The binding of β-catenin to GATA3 promoter is enhanced in primary TEKI MEFs (n = 3 independent experiments). The relative enrichment of β-catenin on the β-catenin binding site of the GATA3 promoter was determined by the ChIP assay as described in Materials and Methods. The binding of GATA3 to its target promoters is increased in primary TEKI MEFs (n = 3 independent experiments). The relative enrichment of GATA3 on the GATA3 binding sites of indicated promoters was determined by the ChIP assay as described in Materials and Methods. Data Information: In (A, B, D, and E), data are presented as mean ± SEM. *P < 0.05 (Mann–Whitney test). Source data are available online for this figure. Source Data for Figure 3 [embr201643803-sup-0007-SDataFig3.pdf] Download figure Download PowerPoint GATA3 has been shown to be highly expressed in preadipocytes, inhibiting adipogenesis in part through repressing the transcription of PPARγ2 2526. In preadipocytes, the transcription of GATA3 is directly activated by itself, as well as by β-catenin, a known SIRT1 deacetylation substrate 2728. The increased expression of GATA3 in TEKI cells suggests that the hyperactive SIRT1 TE protein may induce GATA3 expression through deacetylation/activation of β-catenin. In support of this notion, the binding of β-catenin to the GATA3 promoter was enhanced in these cells despite its reduced protein levels (Fig 3C and D). The association of GATA3 to its own promoter, as well as to PPARγ2 promoter, was also dramatically elevated in TEKI primary MEFs (Fig 3E). These observations suggest that the TE mutant enhances β-catenin/GATA3 pathway thereby inhibiting the induction of PPARγ2 and adipogenesis. In line with the finding in in vitro adipogenesis, β-catenin was significantly hypoacetylated in WAT of TEKI mice compared to WT mice (Fig 4A), suggesting an activation of its transaction activity (Simic et al 27). Consistently, the expression of GATA3 was induced (Fig 4B), whereas the mRNA levels of PPARγ and several PPARγ target genes 29, including Pepck, Adiponectin, aP2, were significantly reduced in the WAT of TEKI mice (Fig 4C). The reduced PPARγ pathway in TEKI WAT was associated with reduced chromatin association of PPARγ to PPAR response element (PPRE) on one of its target promoters, aP2 (Fig 4D). As a control, the binding of PPARγ to a control region on this promoter, C/EBPα binding site (C/EBPα site), was not significantly reduced. Figure 4. SIRT1 TEKI mice have enhanced β-catenin/GATA3 signaling but impaired PPARγ pathway and WAT functions β-Catenin is hypoacetylated in WAT of TEKI mice (n = 4 mice for each group). The acetylation levels of β-catenin were analyzed by immunoprecipitation (IP) of β-catenin followed by immunoblotting (IB) with an anti-acetyl-K antibody. WAT of TEKI mice has increased mRNA levels of GATA3 but not β-catenin (n = 6 mice for each group). The mRNA levels of indicated genes were analyzed by qPCR. TEKI WAT has a reduced PPARγ-signaling pathway (n = 6 mice for each group). The mRNA levels of indicated genes were analyzed by qPCR. Reduced binding of PPARγ on the PPRE of the aP2 promoter in WAT of TEKI mice (n = 3 mice for each group). The relative enrichment of PPARγ on the aP2 promoter was determined by the ChIP assay as described in Materials and Methods. SIRT1 TEKI mice have reduced expression levels of genes involved in lipogenesis in WAT (n = 6 mice for each group). The mRNA levels of indicated genes were analyzed by qPCR. SIRT1 TEKI female mice have reduced body fat under the chow diet feeding (n = 9 WT and 6 TEKI mice). The percentage of fat mass and lean mass in 9- to 10-month-old mice were determined by Bruker LF90 minispec. SIRT1 TEKI mice have reduced expression of genes involved in lipolysis in WAT (n = 6 mice for each group), and SIRT1 TEKI adipocytes have reduced lipolysis in vitro (n = 5 mice for each group). The mRNA levels of indicated genes were analyzed by qPCR, and the in vitro lipolysis assay was performed with isolated primary adipocytes as described in Materials and Methods. SIRT1 TEKI mice have increased expression of genes in energy expenditure in WAT (n = 6 mice for each group). The mRNA levels of indicated genes were analyzed by qPCR. Data Information: In all panels, data are presented as mean ± SEM. 0.05 < #P < 0.1, *P < 0.05 (Mann–Whitney test). Source data are available online for this figure. Source Data for Figure 4 [embr201643803-sup-0008-SDataFig4.pdf] Download figure Download PowerPoint Further analysis revealed that the expression levels of several lipogenic genes were reduced in the WAT of TEKI mice (Fig 4E), and SIRT1 TEKI female mice had significantly reduced fat composition but increased lean mass compared to WT mice (Fig 4F), indicating that TEKI mice have reduced adipogenesis in vivo. Intriguingly, the mRNA levels of a number of genes involved in lipolysis were also significantly reduced in WAT of TEKI mice after the overnight fasting, and isolated primary adipocytes from TEKI mice had reduced release of glycerol in response to the isoproterenol treatment in vitro compared to WT adipocytes (Fig 4G). Moreover, the expression of a couple of genes mediating energy catabolism was also significantly elevated in WAT of TEKI mice (Fig 4E), suggesting that knocking-in the TEKI allele in WAT alters multiple functions of this tissue in addition to adipogenesis/lipogenesis. Together, our findings demonstrate that dephosphorylation of SIRT1 T522 is a critical step for proper differentiation of white adipocytes and that constitutive phosphorylation of SIRT1 at T522 represses white adipocyte differentiation and functions in vitro and in vivo. The SIRT1 TEKI allele enhances systemic lipid oxidation and partially protects mice from high-fat diet-induced dyslipidemia TEKI mice also had elevated serum β-hydroxybutyrate levels after overnight fasting along with enhanced expression of a number of fatty acid oxidation genes in the liver compared to WT control mice (Fig 5A and B), indicating an elevation in hepatic fatty acid oxidation. Moreover, several fatty acid oxidation genes were also significantly increased in the BAT without significant alterations in levels of fatty acid synthesis genes (Fig 5C), further supporting the notion that the TEKI allele enhances systemic fatty acid catabolism. As an additional control, TAKI mice exhibited normal fatty acid oxidation in their liver and BAT (Fig EV2). Figure 5. SIRT1 TEKI mice display enhanced systemic lipid oxidation and are partially protected from high-fat diet-induced dyslipidemia A, B. SIRT1 TEKI mice have increased β-hydroxybutyrate levels in serum (A) and elevated expression levels of fatty acid oxidation genes in the liver (B) in response to fasting (n = 6 WT and 7 TEKI mice). The serum levels of β-hydroxybutyrate was determined as described in Materials and Methods after 16-h fasting, and the mRNA levels of indicated genes were analyzed in fasted liver samples by qPCR. C. SIRT1 TEKI mice have elevated expression of fatty acid oxidation genes in basal condition in the BAT (n = 8 WT and 4 TEKI mice). The mRNA levels of indicated genes were analyzed in BAT samples under fed conditions by qPCR. D, E. SIRT1 TEKI mice have reduced body fat (D), and decreased serum levels of cholesterol, free fatty acids, and glycerol (E) after 5 months of high-fat diet feeding (n = 10 WT and 8 TEKI mice). The percentage of fat mass in indicated mice were determined by Bruker LF90 minispec, and the serum levels of indicated metabolites were measured as described in Materials and Methods. Data Information: In (A–D), data are presented as mean ± SEM. *P < 0.05, **P < 0.01 (Mann–Whitney test). Download figure Download PowerPoint Click here to expand this figure. Figure EV2. SIRT1 TAKI mice have normal fasting-induced hepatic fatty acid oxidation and basal fatty acid oxidation in BAT A, B. SIRT1 TAKI mice have normal levels of serum β-hydroxybutyrate levels (A) and expression of fatty acid oxidation genes in the liver (B) upon fasting (n = 6 mice for each group). C. SIRT1 TAKI mice have normal expression of genes involved in fatty acid metabolism in basal condition in the BAT (n = 4 WT and 8 TAKI mice). Data Information: In all panels, data are presented as mean ± SEM. 0.05 < #P < 0.1 (Mann–Whitney test). Download figure Download PowerPoint To further assess the importance of SIRT1 phosphorylation in control of systemic lipid homeostasis, we challenged WT, TEKI, and TAKI mice with a Western style high-fat diet containing 40% kcal fat and 0.21% cholesterol for 5 months. As shown in Fig 5D, after high-fat diet feeding, TEKI mice displayed a mild but significant reduction in total fat percentage, despite that they gained similar weights (Fig EV3A) and had comparable food intakes (Fig EV3B) compared to WT mice. Moreover, TEKI mice showed decreased serum levels of free fatty acids, glycerol, and cholesterol after high-fat diet feeding (Fig 5E), without detectable alterations in other serum lipids and hormones (Fig EV3E and F). Again, TAKI mice exhibited no significant changes in diet-induced obesity and dyslipidemia (Fig EV3). Collectively, these data indicate that constitutive phosphorylation of SIRT1 on T522 enhances systemic lipid oxidation and partially protects animals from high-fat diet-induced dyslipidemia. Click here to expand this figure. Figure EV3. SIRT1 TEKI and TAKI mice under high-fat diet feeding A, B. Body weight (A) and food intake (B) of SIRT1 TEKI and TAKI mice under high-fat diet feeding (n = 10 WT, 8 TAKI, and 8 TEKI mice). C. SIRT1 TAKI mice have normal total fat percentage after 5 months of high-fat diet feeding (n = 10 WT and 8 TAKI mice). D. SIRT1 TAKI mice have normal serum levels of triglyceride, free fatty acids, glycerol, and cholesterol after 5 months of high-fat diet feeding (n = 10 WT and 8 TAKI mice). E. Serum levels of HDL and LDL after 5 months of high-fat diet feeding (n = 10 WT and 8 TAKI mice). F. Serum insulin, leptin, and adiponectin levels were measured after 5 months of high-fat diet feeding (n = 10 WT and 8 TAKI mice). Data Information: In all panels, data are presented as mean ± SEM. Download figure Download PowerPoint SIRT1 TAKI mice display defective hepatic fatty acid oxidation and develop hepatic steatosis upon high-fat diet feeding In addition to adipose tissues, SIRT1 also has important roles in regulation of hepatic energy metabolism, particularly stimulation of hepatic fatty acid oxidation 678. But again, how the activity of hepatic SIRT1 is regulated in response to nutritional cues is still unclear. To explore the possible role of T522 phosphorylation in regulation of SIRT1 activity in liver, we analyzed the hepatic pSIRT1 (T522) levels in ch" @default.
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• W2598459868 title "The phosphorylation status of T522 modulates tissue‐specific functions of <scp>SIRT</scp> 1 in energy metabolism in mice" @default.
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