The phosphorylation status of T522 modulates tissue-specific functions of SIRT1 in energy metabolism in mice

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1 Published online: March, 7 Article The phosphorylation status of T modulates tissue-specific functions of SIRT in energy metabolism in mice Jing Lu,,, Qing Xu,, Ming Ji,, Xiumei Guo,,, Xiaojiang Xu, David C Fargo & Xiaoling Li, Abstract SIRT, the most conserved mammalian NAD + -dependent protein deacetylase, is an important metabolic regulator. However, the mechanisms by which SIRT is regulated in vivo remain unclear. Here, we report that phosphorylation modification of T on SIRT is crucial for tissue-specific regulation of SIRT activity in mice. Dephosphorylation of T is critical for repression of its activity during adipogenesis. The phospho-t level is reduced during adipogenesis. Knocking-in a constitutive T phosphorylation mimic activates the b-catenin/gata pathway, repressing PPARc signaling, impairing differentiation of white adipocytes, and ameliorating high-fat diet-induced dyslipidemia in mice. In contrast, phosphorylation of T is crucial for activation of hepatic SIRT in response to over-nutrition. Hepatic SIRT is hyperphosphorylated at T upon high-fat diet feeding. Knocking-in a SIRT mutant defective in T phosphorylation disrupts hepatic fatty acid oxidation, resulting in hepatic steatosis after high-fat diet feeding. In addition, the T dephosphorylation mimic impairs systemic energy metabolism. Our findings unveil an important link between environmental cues, SIRT phosphorylation, and energy homeostasis and demonstrate that the phosphorylation of T is a critical element in tissue-specific regulation of SIRT activity in vivo. Keywords adipogenesis; hepatic steatosis; liver damage; phosphorylation; SIRT Subject Categories Metabolism; Post-translational Modifications, Proteolysis & Proteomics DOI./embr.648 Received December 6 Revised February 7 Accepted February 7 Introduction Metabolic syndrome is defined as a cluster of metabolism-related disorders, such as central obesity, type diabetes, dyslipidemia, and high blood pressure, all of which are considered as major contributors of mortality in industrialized countries [ 4]. 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 (Sir) protein, SIRT, play a central role in the regulation of transcriptional networks in various critical metabolic processes in multiple tissues. For example, SIRT is a key modulator of both glucose and fatty acid metabolism in the liver [,6]. Knocking-down or deletion of hepatic SIRT impairs fatty acid oxidation, thereby increasing the susceptibility of mice to dyslipidemia and hepatic steatosis [7 9]. Conversely, hepatic overexpression of SIRT attenuates hepatic steatosis and ER stress and restores glucose homeostasis in mice []. SIRT is also an important regulator of maturation and remodeling of adipose tissues [6]. It has been reported that SIRT represses a master regulator of adipogenesis in the white adipose tissue (WAT), PPARc, thereby suppressing the expression of adipose tissue markers, such as a fatty acid binding protein, ap, and inhibiting fat mobilization in response to fasting []. Moreover, genetic ablation of SIRT in adipose tissues leads to increased adiposity and insulin resistance [], whereas treatment of mice on a high-fat diet with resveratrol, a polyphenol that activates SIRT in cells directly or indirectly [ 7], protects animals against high-fat induced obesity and metabolic dysfunctions [8 ]. Therefore, current studies point to the notion that SIRT 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 []. Although the role of SIRT in metabolic regulation of a variety of biological processes has been well studied, how the activity of SIRT is regulated in vivo in response to different biological/environmental cues remains elusive, and the functional/physiological 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 Integrative Bioinformatics, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA Corresponding author. Tel: ; lix@niehs.nih.gov These authors contributed equally to this work Present address: Guangzhou DiagCor Clinical Laboratory Co., Ltd, Guangzhou, Guangdong, China Published 7. This article is a U.S. Government work and is in the public domain in the USA

2 Published online: March, 7 Tissue-specific SIRT phosphorylation Jing Lu et al consequences of disruption of its regulation are still unclear. As a NAD + -dependent protein deacetylase, the activity of SIRT 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 []. In particular, we have previously reported that SIRT can be phosphorylated and activated by two anti-apoptotic members of the dual-specificity tyrosine phosphorylation-regulated kinase (DYRK), DYRKA and DYRK, in response to acute environmental stresses []. We further showed that this modification activates its deacetylase activity independently of the cellular NAD + level through preventing the formation of less-active SIRT oligomers/aggregates []. Our findings suggest that phosphorylation modification of SIRT might provide a molecular mechanism that fine-tunes SIRT activity in vivo independently of the cellular NAD + level. To assess the physiological impacts of the phosphorylation of T on SIRT, we generated SIRT T phosphorylation mimic (threonine to glutamic acid, or TE mutation, or TE) and dephosphorylation mimic (threonine to alanine, or TA mutation, or TA) knock-in mouse models. In this report, we show for the first time that the phosphorylation of T renders tissue-specific regulation of SIRT activity in response to developmental and nutritional signals in vivo. Results Generation of SIRT and TAKI mice To investigate whether phosphorylation of T of SIRT is an important posttranslational modification that modulates SIRT activity in vivo, we used standard gene-targeting technology to generate two different SIRT mutation knock-in mouse lines, SIRT TAKI (TAKI) and SIRT (), with TA (TA) to mimic the dephosphorylated SIRT and TE (TE) to mimic the phosphorylated SIRT, respectively (Figs and EV). Both KI strains were born at the expected Mendelian ratio with no gross phenotypes, indicating that the phosphorylation status of T does not affect normal animal development and survival. Immunoblotting analysis of total protein lysates from different tissues with anti-sirt antibody revealed that the protein levels of two mutant proteins in these two lines were comparable to those of wild-type () SIRT (Fig A). Further analyses with the antip-sirt(t) antibody confirmed that knocking-in the TA mutant abolished the phosphorylation signals on the endogenous SIRT protein (Fig B, Lane TAKI). Since our p-sirt(t) antibody only binds weakly to the phospho-mimics [], knocking-in the TE mutation also yielded a mutant SIRT protein undetectable by this antibody (Fig B, Lane ). Our previous studies have demonstrated that in cultured cells, the phospho-mimics of SIRT T are hyperactive toward acetylp protein upon genotoxic stress, whereas the dephospho-mimics of SIRT T are partially inactive [,]. To confirm the changes of SIRT deacetylase activity in the knock-in mouse lines, we isolated mouse embryonic fibroblasts (MEFs) from, TAKI, and embryos and analyzed cellular SIRT activities. As shown in Fig C, acetyl-p levels in MEFs were increased when treated with a DNA intercalating drug, adriamycin (Adria). This increase was blunted in MEFs (top panels), indicating that the TE mutant protein has an increased deacetylase activity toward acetylp. The knocked-in SIRT TA protein, on the other hand, did not appear to have defects in deacetylation of p protein in response to adriamycin-induced genotoxic stress (bottom panels), possibly due to stable knock-in induced compensatory effects in regulating p acetylation in these cells. However, both TE and TA mutants displayed expected enhanced (, top panels) and reduced (TAKI, bottom panels) deacetylase activities toward acetyl-p6 when MEFs were challenged with pro-inflammatory stimuli, TNFa or LPS (Fig D). Taken together, our observations indicate that when stably knocked-in mice, the SIRT 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 SIRT at T inhibits adipogenesis in vitro SIRT 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 T plays a role in regulation of SIRT activity in this process, we analyzed the p-sirt(t) levels of endogenous SIRT protein during a 7-day in vitro adipogenesis of primary MEFs. As shown in Fig A, p-sirt levels were gradually reduced while total SIRT protein remained constant during in vitro adipogenesis, indicating that SIRT phosphorylation (activity) but not SIRT expression is negatively correlated with adipogenesis. Moreover, this reduction in p-sirt levels was accompanied with decreased levels of DYRK but not DYRKA (Fig A and B), suggesting that reduced expression of DYRK might be a reason for the diminished phosphorylation of SIRT in this process. In line with above observations, primary MEFs isolated from dephosphorylation defective mice accumulated much less fat as determined by Oil Red O staining after 7 days of differentiation compared with MEFs, whereas MEFs from constitutively dephosphorylated TAKI mice displayed a comparable ability to be differentiated into adipocytes as MEFs (Fig C). Consistently, the mrna levels of PPARc, a nutrition-sensitive isoform of PPARc, ap, as well as Glut4, an adipose tissue-specific glucose transporter, were significantly reduced in but not in TAKI cells during differentiation (Fig D). Taken together, our finding indicates that dephosphorylation of SIRT, thereby reduction in SIRT activity, is required for normal adipogenesis in vitro. The SIRT allele actives b-catenin/gata signaling, repressing PPARc and impairing functions of WAT in vitro and in vivo Although SIRT has been shown to indirectly repress or directly activate PPARc in response to different environmental signals in WAT [,4], the reduced induction of PPARc during adipogenesis of cells (Fig D) raises the possibility that the SIRT TE mutant protein may blunt the transcription of PPARc, thereby repressing adipocyte differentiation. To test this possibility, we analyzed the expression of several genes that have been previously shown to be Published 7. This article is a U.S. Government work and is in the public domain in the USA

3 Published online: March, 7 Jing Lu et al Tissue-specific SIRT phosphorylation A TAKI B TAKI SIRT β-actin Testis p-sirt SIRT Testis SIRT β-actin Liver p-sirt SIRT Liver SIRT β-actin WAT p-sirt SIRT WAT p-sirt SIRT BAT C D DMSO Adria DMSO Adria acetylp p acetylp6 p6 PBS TNFα LPS PBS TNFα LPS TAKI TAKI acetylp DMSO Adria DMSO Adria acetylp6 PBS TNFα LPS PBS TNFα LPS p p6 Figure. Generation of SIRT and TAKI mice. A The SIRT expression levels in wild type (),, and TAKI mice are comparable. Total protein lysates from indicated tissues were analyzed by immunoblotting with an anti-sirt antibody. B Endogenous SIRT proteins from indicated tissues from both and TAKI mice display decreased p-sirt(t) levels. Total protein lysates from indicated tissues were analyzed by immunoblotting with antibodies against SIRT and p-sirt(t). Please note that the p-sirt(t) antibody only displays weak affinity to the SIRTTE protein. C SIRT MEFs have an increased deacetylase activity toward p in response to genotoxic stress. MEFs isolated from,, and TAKI mice were treated with adriamycin (. lg/ml) for 8 h or with PBS (as control). Total cell lysates were immunoblotted with acetyl-p (K8) orp antibodies. D SIRT MEFs have elevated deacetylase activity to the p6 subunit of NF-jB in response to inflammatory signals.,, and TAKI MEFs were treated with ng/ml of TNFa, or lg/ml of Escherichia coli O:B4 lipopolysaccharide (LPS) for min with PBS (as control). Total cell lysates were immunoblotted with acetylp6 and p6 antibodies. Source data are available online for this figure. important in transcriptional regulation of PPARc, such as b-catenin, GATA, and GATA, in and MEFs before and after differentiation into adipocytes. As shown in Fig A C, both mrna and protein levels of GATA but not its close homolog GATA nor b-catenin were significantly elevated in MEFs before and at the early stages of differentiation. Published 7. This article is a U.S. Government work and is in the public domain in the USA

4 Published online: March, 7 Tissue-specific SIRT phosphorylation Jing Lu et al A Days after differentiation p-sirt SIRT DYRK β-actin B Dyrka Dyrk Days after differentiation C TAKI D Pparγ 6 C/ebpα ap Glut4 7 Days after differentiation Figure. The SIRT allele inhibits adipogenesis in vitro. A The endogenous SIRT protein is dephosphorylated at T during in vitro differentiation of MEFs into adipocytes. Primary MEFs isolated from 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. B The expression levels of Dyrk but not Dyrka are decreased during in vitro adipogenesis (n = independent experiments). The mrna levels of indicated genes were analyzed by qpcr. C SIRT MEFs have reduced in vitro adipogenesis. In vitro differentiated adipocytes from primary MEFs isolated from, TAKI, and mice were stained by Oil Red O. Scale bars, lm. D In vitro differentiated adipocytes from primary MEFs have reduced expression levels of adipocyte markers (n = independent experiments). Data Information: In (B and D), data are presented as mean SEM. P <. (Mann Whitney test). Source data are available online for this figure. 4 Published 7. This article is a U.S. Government work and is in the public domain in the USA

5 Published online: March, 7 Jing Lu et al Tissue-specific SIRT phosphorylation A.. β-catenin Gata B 8 4. Days after differentiation C β-catenin GATA SIRT β-actin D Relative Enrichment ChIP: GATA β-catenin site E Relative Enrichment ChIP: GATA GATA site PPARγ GATA site Figure. The SIRT allele actives b-catenin/gata signaling. A MEFs have increased expression levels of GATA during in vitro adipogenesis (n = independent experiments). The mrna levels of indicated genes were analyzed by qpcr. B Primary MEFs have increased mrna levels of GATA but not b-catenin (n = 4 independent experiments). The mrna levels of indicated genes were analyzed by qpcr. C Primary MEFs have increased protein levels of GATA but reduced b-catenin protein. The levels of indicated protein were analyzed by immunoblotting. D The binding of b-catenin to GATA promoter is enhanced in primary MEFs (n = independent experiments). The relative enrichment of b-catenin on the b-catenin binding site of the GATA promoter was determined by the ChIP assay as described in Materials and Methods. E The binding of GATA to its target promoters is increased in primary MEFs (n = independent experiments). The relative enrichment of GATA on the GATA 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 <. (Mann Whitney test). Source data are available online for this figure. GATA has been shown to be highly expressed in preadipocytes, inhibiting adipogenesis in part through repressing the transcription of PPARc [,6]. In preadipocytes, the transcription of GATA is directly activated by itself, as well as by b-catenin, a known SIRT deacetylation substrate [7,8]. The increased expression of GATA in cells suggests that the hyperactive SIRT TE protein may induce GATA expression through deacetylation/activation of b-catenin. In support of this notion, the binding of b-catenin to the GATA promoter was enhanced in these cells despite its reduced protein levels (Fig C and D). The association of GATA to its own promoter, as well as to PPARc promoter, was also dramatically elevated in primary MEFs (Fig E). These observations suggest that the TE mutant enhances b-catenin/gata pathway thereby inhibiting the induction of PPARc and adipogenesis. In line with the finding in in vitro adipogenesis, b-catenin was significantly hypoacetylated in WAT of mice compared to mice (Fig 4A), suggesting an activation of its transaction activity (Simic et al [7]). Consistently, the expression of GATA was induced (Fig 4B), whereas the mrna levels of PPARc and several PPARc target genes [9], including Pepck, Adiponectin, ap, were significantly reduced in the WAT of mice (Fig 4C). The reduced PPARc pathway in WAT was associated with reduced chromatin association of PPARc to PPAR response element (PPRE) on one of its target promoters, ap (Fig 4D). As a control, the Published 7. This article is a U.S. Government work and is in the public domain in the USA

6 Published online: March, 7 Tissue-specific SIRT phosphorylation Jing Lu et al A acetylβ-catenin β-catenin β-catenin β-actin IP:β-Catenin; IB: acetyl-k IP:β-Catenin; IB: β-catenin Input Input Relative Acetyl- /total β-catenin B... C PPARγ pathway D ap PPRE ap C/EBPα site..8.4 # Relative Enrichment ChIP: E Lipogenesis F Body weight (g) Body weight Fat mass Lean mass Fat weight % Lean mass % G Lipolysis Release glycerol (μmol/h/μg DNA) 9 6 in vitro lipolysis H Catabolism Figure 4. 6 Published 7. This article is a U.S. Government work and is in the public domain in the USA

7 Published online: March, 7 Jing Lu et al Tissue-specific SIRT phosphorylation Figure 4. SIRT mice have enhanced b-catenin/gata signaling but impaired PPARc pathway and WAT functions. A b-catenin is hypoacetylated in WAT of mice (n = 4 mice for each group). The acetylation levels of b-catenin were analyzed by immunoprecipitation (IP) of b- catenin followed by immunoblotting (IB) with an anti-acetyl-k antibody. B WAT of mice has increased mrna levels of GATA but not b-catenin (n = 6 mice for each group). The mrna levels of indicated genes were analyzed by qpcr. C WAT has a reduced PPARc-signaling pathway (n = 6 mice for each group). The mrna levels of indicated genes were analyzed by qpcr. D Reduced binding of PPARc on the PPRE of the ap promoter in WAT of mice (n = mice for each group). The relative enrichment of PPARc on the ap promoter was determined by the ChIP assay as described in Materials and Methods. E SIRT 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. F SIRT female mice have reduced body fat under the chow diet feeding (n = 9 and 6 mice). The percentage of fat mass and lean mass in 9- to- month-old mice were determined by Bruker LF9 minispec. G SIRT mice have reduced expression of genes involved in lipolysis in WAT (n = 6 mice for each group), and SIRT adipocytes have reduced lipolysis in vitro (n = 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. H SIRT 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.. < # P <., P <. (Mann Whitney test). Source data are available online for this figure. binding of PPARc to a control region on this promoter, C/EBPa binding site (C/EBPa site), was not significantly reduced. Further analysis revealed that the expression levels of several lipogenic genes were reduced in the WAT of mice (Fig 4E), and SIRT female mice had significantly reduced fat composition but increased lean mass compared to mice (Fig 4F), indicating that 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 mice after the overnight fasting, and isolated primary adipocytes from mice had reduced release of glycerol in response to the isoproterenol treatment in vitro compared to adipocytes (Fig 4G). Moreover, the expression of a couple of genes mediating energy catabolism was also significantly elevated in WAT of mice (Fig 4E), suggesting that knocking-in the allele in WAT alters multiple functions of this tissue in addition to adipogenesis/lipogenesis. Together, our findings demonstrate that dephosphorylation of SIRT T is a critical step for proper differentiation of white adipocytes and that constitutive phosphorylation of SIRT at T represses white adipocyte differentiation and functions in vitro and in vivo. The SIRT allele enhances systemic lipid oxidation and partially protects mice from high-fat diet-induced dyslipidemia mice also had elevated serum b-hydroxybutyrate levels after overnight fasting along with enhanced expression of a number of fatty acid oxidation genes in the liver compared to control mice (Fig A 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 C), further supporting the notion that the allele enhances systemic fatty acid catabolism. As an additional control, TAKI mice exhibited normal fatty acid oxidation in their liver and BAT (Fig EV). To further assess the importance of SIRT phosphorylation in control of systemic lipid homeostasis, we challenged,, and TAKI mice with a Western style high-fat diet containing 4% kcal fat and.% cholesterol for months. As shown in Fig D, after high-fat diet feeding, mice displayed a mild but significant reduction in total fat percentage, despite that they gained similar weights (Fig EVA) and had comparable food intakes (Fig EVB) compared to mice. Moreover, mice showed decreased serum levels of free fatty acids, glycerol, and cholesterol after highfat diet feeding (Fig E), without detectable alterations in other serum lipids and hormones (Fig EVE and F). Again, TAKI mice exhibited no significant changes in diet-induced obesity and dyslipidemia (Fig EV). Collectively, these data indicate that constitutive phosphorylation of SIRT on T enhances systemic lipid oxidation and partially protects animals from high-fat diet-induced dyslipidemia. SIRT TAKI mice display defective hepatic fatty acid oxidation and develop hepatic steatosis upon high-fat diet feeding In addition to adipose tissues, SIRT also has important roles in regulation of hepatic energy metabolism, particularly stimulation of hepatic fatty acid oxidation [6 8]. But again, how the activity of hepatic SIRT is regulated in response to nutritional cues is still unclear. To explore the possible role of T phosphorylation in regulation of SIRT activity in liver, we analyzed the hepatic psirt (T) levels in chow diet and high-fat diet-fed mice either under fed condition or after overnight fasting (Fig 6A and B). Surprisingly, compared to chow diet-fed livers, p-t levels were significantly induced in high-fat diet-fed livers but not upon fasting. Since cellular NAD + levels have been reported to be reduced after high-fat diet feeding [,], this observation suggests that SIRT activity is increased upon over-nutrition independently of cellular NAD + levels and further suggests that TAKI mice, in which SIRT is defective in T phosphorylation, would be more sensitive to lipid loading than and mice in the liver. In agreement with this hypothesis, TAKI mice exhibited significant reduction in several fatty acid oxidation genes in the liver under the fed condition compared to and mice (Fig 6C). Moreover, TAKI primary hepatocytes showed a blunted ability to induce the expression of a number of fatty acid oxidation genes in response to lipid loading followed by the treatment with a PPARa agonist, WY464 (WY; Fig 6D), indicating that the TA mutant decreases fatty acid oxidation cell autonomously in hepatocytes. On the other hand, although mice had enhanced hepatic fatty acid oxidation in response to fasting (Fig A and B), the isolated primary hepatocytes from these Published 7. This article is a U.S. Government work and is in the public domain in the USA 7

8 Published online: March, 7 Tissue-specific SIRT phosphorylation Jing Lu et al A Serum B Liver (fasted) β-hydroxybutyrate (mm) C BAT (fed).. Oxidation Synthesis Others D Fat weight % 4 E Serum Triglyceride (mg/ml) Triglyceride NEFA (mm) NEFA Glycerol (mg/ml) Glycerol Cholesterol (mg/dl) Cholesterol Figure. SIRT mice display enhanced systemic lipid oxidation and are partially protected from high-fat diet-induced dyslipidemia. A, B SIRT mice have increased b-hydroxybutyrate levels in serum (A) and elevated expression levels of fatty acid oxidation genes in the liver (B) in response to fasting (n = 6 and 7 mice). The serum levels of b-hydroxybutyrate was determined as described in Materials and Methods after 6-h fasting, and the mrna levels of indicated genes were analyzed in fasted liver samples by qpcr. C SIRT mice have elevated expression of fatty acid oxidation genes in basal condition in the BAT (n = 8 and 4 mice). The mrna levels of indicated genes were analyzed in BAT samples under fed conditions by qpcr. D, E SIRT mice have reduced body fat (D), and decreased serum levels of cholesterol, free fatty acids, and glycerol (E) after months of high-fat diet feeding (n = and 8 mice). The percentage of fat mass in indicated mice were determined by Bruker LF9 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 <., P <. (Mann Whitney test). 8 Published 7. This article is a U.S. Government work and is in the public domain in the USA

9 Published online: March, 7 Jing Lu et al Tissue-specific SIRT phosphorylation A Liver p-sirt SIRT Fed Chow Fasted Chow Fed HFD B Liver Relative amount of p-srt/sirt C Liver (fed) Acad Ehhadh Cd6 Cyp4a Fgf Mcad. Chow HFD TAKI D Primary Hepatocytes. Acad 8 Ehhadh Aox Cpta Cd6 8 Acot... # 6.. # # DMSO WY TAKI DMSO TAKI WY E Primary hepatocytes Acad Ehhadh Aox Cpta Cd6 Acot DMSO WY DMSO WY Figure 6. Published 7. This article is a U.S. Government work and is in the public domain in the USA 9

10 Published online: March, 7 Tissue-specific SIRT phosphorylation Jing Lu et al Figure 6. SIRT TAKI mice have impaired hepatic fatty acid oxidation under fed or lipid-loading conditions. A T phosphorylation levels on endogenous SIRT protein are increased in high-fat diet-fed livers. mice were fed with a chow diet (Chow) or a high-fat diet (HFD) for months. Liver p-sirt (T) and total SIRT levels were analyzed by immunoblotting under indicated conditions. B p-sirt (T) levels in livers of mice under different conditions (n = mice for each group). The relative levels of p-sirt/total SIRT were determined by densitometry with ImageJ. C SIRT TAKI mice have reduced hepatic expression of genes involved in fatty acid oxidation under fed condition (n = 6 mice for each group). The mrna levels of indicated genes were analyzed in liver samples under fed conditions by qpcr. D, E SIRT TAKI but not primary hepatocytes are defective in fatty acid oxidation upon lipid loading and treatment with a PPARa agonist, WY464 (WY; n = 4 mice for each group). Primary hepatocytes isolated from,, or TAKI mice were pre-incubated with lm oleic acid/bsa for overnight in the presence or absence of lm WY464 in low glucose medium, followed by incubation with lm oleic acid/bsa and mm carnitine in glucose-free DMEM for 4 h. Data Information: In (B E), data are presented as mean SEM.. < # P <., P <., P <. (Mann Whitney test). Source data are available online for this figure. mice had a normal ability to induce the expression of fatty acid oxidation genes after lipid loading and WY treatment (Fig 6E), suggesting that the elevation in hepatic fatty acid oxidation in mice is non-cell autonomous. Consistent with above observations, TAKI mice accumulated significantly higher amount of fat in their liver after months of high-fat diet feeding, as revealed by H&E staining of liver sections (Fig 7A) and enzymatic colorimetric quantification of extracted liver triglycerides, NEFA, and cholesterol (Fig 7B). Further analyses showed that after months of high-fat diet feeding, TAKI livers also had enhanced deposition of collagen (Fig 7C, arrows), hepatocyte nuclear invagination and enlarged nuclei (Fig 7D, arrows), and increased serum levels of AST (Fig 7E), indicating development of liver fibrosis and liver damage. Further microarray analysis of hepatic gene expression of highfat diet-fed, TAKI, and mice revealed that consistent with above observations, and livers shared almost identical gene expression profiles under the high-fat diet (Fig 7F). On the contrary, TAKI livers had alterations on the expression of,86 and,46 gene probes compared to and livers, respectively (Fig 7F, top, Tables EV and EV), and 866 of these genes were common (Fig 7F, bottom), suggesting that TAKI livers were significantly different from and livers. In support of this notion, heat map analyses of total significantly changed,66 gene probes indicated that and liver were indistinguishable, whereas TAKI livers were clustered into a separate group (Fig EV4). Further Ingenuity Pathway Analysis (IPA) of these,66 genes revealed that TAKI livers had significantly impairments on pathways involved in hepatic fibrosis/hepatic stellate cell activation (Table EV) as well as the liver toxicity and inflammation (Fig 7G and H) compared to and livers, confirming that SIRT phosphorylation on T is crucial to maintain hepatic functions in response to nutrient overloading. Collectively, our findings indicate that phosphorylation of the T residue on SIRT is vital for hepatic fatty acid oxidation upon high-fat diet feeding. SIRT TAKI mice exhibit systemic alternations in energy metabolism Our observation that phosphorylation modification of SIRT T was widespread in all tested tissues (Fig B) further suggests that knocking-in a constitutive dephosphorylated SIRT allele may elicit systemic effects on SIRT-mediated metabolic processes in addition to lipid-induced hepatic fatty acid oxidation. Indeed, SIRT TAKI mice were insulin resistant under normal feeding conditions (Fig 8A), indicating that they are less able to uptake glucose. On the contrary, SIRT mice have normal insulin sensitivity (Fig 8B). Paradoxically, TAKI mice also exhibited an enhanced respiration exchange ratio (RER) compared to and mice, especially at the night phase (Fig 8C and D), indicating a preference to use glucose over fatty acids of these mice. One plausible explanation for this paradox is that the blunted ability to utilize fat (Figs 6 and 7) increases the glucose dependence of TAKI mice in spite of the fact that they are less able to uptake glucose in response to insulin. Taken together, our studies demonstrate that the phosphorylation modification of the SIRT T residue is critically involved in Figure 7. SIRT TAKI mice develop fatty liver after high-fat diet feeding. A SIRT TAKI mice develop fatty liver after months of high-fat diet feeding. Liver sections were stained with hematoxylin and eosin. Scale bars, lm. B TAKI mice have increased triglyceride and non-esterified fatty acids (NEFAs) in the liver after high-fat diet feeding (n = 7 mice for each group). The hepatic lipids metabolites were extracted and measured as described in Materials and Methods. C SIRT TAKI mice have increased liver fibrosis after high-fat diet feeding. Liver sections were stained by Masson s trichrome staining (blue). Scale bars, lm. D Hepatocyte nuclear invagination and enlarged nuclei (arrows) in TAKI liver after high-fat diet feeding. Scale bar, lm. E TAKI mice have enhanced liver damage after high-fat diet feeding. Serum AST and ALT levels were measured after months of high-fat diet feeding (n = 7 mice for each group). F TAKI mice have a distinct hepatic gene expression profile compared to and mice. (Top) The numbers of differentially expressed gene probes between, TAKI, and livers. (Bottom) Venn-diagram representation of significantly altered gene probes between TAKI vs. and TAKI vs.. The hepatic mrna was analyzed by mouse whole-genome microarray as described in Materials and Methods. G SIRT TAKI livers have differential expression patterns of a subset of genes in the liver toxicity and inflammation pathways compared to and livers (n = mice, TAKI mice, and mice; cutoff P <.). The hepatic mrna was analyzed by mouse whole-genome microarray as described in Materials and Methods. H SIRT TAKI livers have increased expression of several genes involved in liver toxicity and inflammation (n = mice and TAKI mice). The hepatic mrna was analyzed by quantitative real-time PCR. Data Information: In (B, E and H), data are presented as mean SEM.. < # P <., P <., P <. (Mann Whitney test). Published 7. This article is a U.S. Government work and is in the public domain in the USA

11 Published online: March, 7 Jing Lu et al Tissue-specific SIRT phosphorylation A TAKI E AST (u/l) AST B Triglyceride (mg/gliver) Triglyceride NEFA (mmol/gliver) 4 NEFA Cholesterol (mg/gliver) Cholesterol ALT (u/l) ALT # C D TAKI TAKI F 86 G TA 46 TE Il Cd44 Cd4 Il4 H TA vs TA vs TE TAKI # Col4a6 Myh4 Tnfrsfb Myh Errb Il Errb Il6 - - Figure 7. Published 7. This article is a U.S. Government work and is in the public domain in the USA

12 Published online: March, 7 Tissue-specific SIRT phosphorylation Jing Lu et al A Glucose (mg/dl) TA TAKI Time after injection (min) B TE Time after injection (min) C D.. Respiratory Exchange Ratio Night Day Night Day Night Day Night Day Night Day Night Day TAKI Figure 8. SIRT TAKI mice display systemic alterations in energy metabolism. A SIRT TAKI mice are more insulin resistant than mice under normal feeding conditions. Six hours after fasting,, SIRT TAKI, and SIRT mice were i.p. injected with.7 l/kg insulin, and blood glucose levels were monitored (n = 7 and 8 TAKI littermate mice). B SIRT mice display a normal sensitivity to insulin compared to controls under normal feeding conditions (n = 7 and 8 littermate mice). C, D SIRT TAKI mice have increased respiratory exchange ratio (RER) during the night phase, whereas SIRT mice display normal levels of RER., TAKI, and mice were singly housed in the Labmaster calorimetry units, and the rates of O consumption and CO production were monitored and the RER = VCO /VO (n = 7 and eight TAKI littermate mice or n = 7 and eight littermate mice). Data Information: In (A and B), data are presented as mean SEM. P <. (Mann Whitney test). In (C and D), data are presented as mean. normal function of SIRT in systemic energy metabolism in response to nutritional signals in vitro and in vivo. Discussion As a critical cellular metabolic sensor, SIRT has been well established as a master regulator of metabolism [6,,]. However, the regulation of SIRT activity in vivo in response to environmental signals is still unclear, and the physiological consequences of disruption of its regulation are largely unknown. In our present study, we showed that the phosphorylation modification of SIRT on T is an important regulatory mechanism for modulation of the activity of SIRT in energy metabolism in different tissues. On the one hand, an allele that mimics the phosphorylation of T, the allele, primarily affects maturation and function of WAT. One the other hand, a SIRT mutant allele that is defective in phosphorylation modification, the TAKI allele, disrupts systemic energy homeostasis, such as hepatic fatty acid metabolism, insulin sensitivity, and circadian switch of nutrient resources. Our observations suggest that maintaining a proper phosphorylation level of T is critical for whole body energy homeostasis, while Published 7. This article is a U.S. Government work and is in the public domain in the USA

13 Published online: March, 7 Jing Lu et al Tissue-specific SIRT phosphorylation dephosphorylation of T is an important regulatory mechanism to repress SIRT activity during adipogenesis. Our study highlights the importance of SIRT T phosphorylation in coupling environmental cues to energy metabolism in vivo. Several lines of evidence support the notion that constitutive phosphorylation of T (the TE mutation) primarily impacts the function of SIRT in adipose tissues. For example, SIRT primary MEFs display defective adipogenesis in vitro (Fig ). SIRT mice have a blunted fasting response in WAT, enhanced energy expenditure in BAT, and are relatively protected from high-fat diet-induced dyslipidemia (Figs 4 and ). More importantly, the T residue of endogenous SIRT protein is gradually dephosphorylated during the process of in vitro adipogenesis (Fig A), indicating that the dephosphorylation of T is a critical mechanism in repression of SIRT activity in this process. Despite the strong influence of the allele on adipose tissues, however, the impact of this allele on functions of other tissues appears to be minimal. For instance, the allele only indirectly enhances hepatic fatty acid oxidation in response to fasting (Figs A and B, and 6E), and SIRT mice and controls have comparable hepatic responses to high-fat diet feeding (Fig 7). SIRT mice also have normal expression levels of genes involved in lipid metabolism in the muscle (Fig EV) and display normal insulin sensitivity (Fig 8B) and a normal nutrient preference (Fig 8D) under normal feeding conditions when compared with mice. In addition to inhibition of the PPARc signaling (Figs 4 and []), SIRT has been shown to directly enhance this signaling through deacetylation of PPARc itself in response to cold exposure in the WAT, thereby promoting WAT browning [4]. Therefore, the adipose tissue specificity of mice raises the possibility that they may have enhanced cold-induced browning of WAT. It will be interesting to test this possibility in future studies. Despite the strong defects of TAKI mice in hepatic lipid metabolism upon high-fat diet feeding (Figs 6 and 7), the molecular targets of the SIRT TAKI mutant protein in liver are still not clear. We have examined the acetylation status of a number of known SIRT targets, including PGC-a, FOXO, NF-kB (p6), p, and H4K6, in livers of high-fat diet-fed animals, but none of these proteins displayed altered acetylation levels in the liver of TAKI mice compared to mice (Fig EV6). New assays are needed to identify possible targets of SIRT in liver of TAKI mice in future studies. The tissue-specific impact of T phosphorylation on SIRT s function is not altogether surprising. Although the SIRT T residue is phosphorylated in all tested tissues (Fig B, not shown), the expression of DYRK kinases is tissue-specific [4,]. In particular, the expression levels of several DYRKs, including DYRK and DYRKB, are highly sensitive to environmental stress and nutritional cues [,6,7]. Additionally, the T of human SIRT (equivalent to T of mouse SIRT) has been reported also as a target of cyclin B/cdk and JNK kinase [8,9]. Therefore, the phosphorylation level of SIRT is differentially regulated by different kinases in different tissues in response to different stimuli. Our observations that the TAKI allele behaves like the allele in tissues/conditions where the endogenous SIRT is hypophosphorylated (e.g., adipogenesis), while the allele behaves like the allele in tissues/conditions where the endogenous SIRT is hyperphosphorylated (e.g., HFD-fed liver), are consistent with this notion. Despite the tissue specificity of T phosphorylation, however, our observations that TAKI mice have defects in multiple tissues support the idea that endogenous SIRT protein is generally phosphorylated at T in different tissues, and maintaining this basal phosphorylation level of T is critical for whole body energy homeostasis. Our observation that T was hyperphosphorylated in high-fat diet-fed livers in which cellular NAD + levels are reduced [,] further demonstrates that the activity of SIRT can be induced independently of cellular NAD + level in vivo. The link between DYRK and SIRT suggests that DYRK kinases may be also vital in regulation of energy metabolism in addition to neuronal development [4], stress response [4], and cell proliferation and apoptosis [4,4,4]. Indeed, the essential role of DYRKs in energy metabolism has begun to emerge in recent years. For instance, minibrain/dyrka has recently been shown to regulate food intake in flies and mammals through the Sir-FOXO-sNPF/NPY pathway [44], confirming our link between DYRKA and SIRT in this process []. DYRKA has also been reported to induce pancreatic b-cell mass expansion and improve glucose tolerance in mice [4,46]. Moreover, DYRKB, a close member of DYRKA, has been recently associated with a form of the metabolic syndrome in human [47]. Our observation that the reduced expression of DYRK is coupled with decreased SIRT phosphorylation during adipogenesis (Fig A and B) further suggests that this kinase may be also involved in energy metabolism in mammals. Further studies with genetic modified mouse models will help to test this possibility in vivo. It is important to note that although the phosphorylation defective TAKI allele displays a number of expected alterations in hepatic and systemic metabolic homeostasis, some of our current observations are inconsistent with our previous results or expected phenotypes. For instance, we have previously shown that in cultured cells and in vitro, the phosphorylation defective TA mutant protein is aggregation prone and less active, failing to protect cells from stress-induced cell death [,]. Given the strong impact of the TA mutation on SIRT activity in vitro, as well as the importance of SIRT activity in animal development [48 ], it is unexpected that the TAKI allele does not significantly affect the deacetylase activity of SIRT in some tested substrates (Fig D) and the TAKI mice have minimal alterations on development (not shown). Since dephosphorylation of SIRT T primarily affects the stability of SIRT protein instead of its intrinsic deacetylase activity [,], it is possible that compensatory effects induced by stable knocking-in the TA mutant, including hyperphosphorylation of SIRT on additional sites, offset some effects of the TA mutation on SIRT oligomerization/aggregation. Nevertheless, our data indicate that maintaining a suitable phosphorylation level of T on SIRT protein is critical for energy homeostasis in multiple tissues, highlighting the importance of this modification in vivo. In summary, we have shown that phosphorylation modification of T is an additional layer that mediates environmental modulation of SIRT activity in vivo, and plays a vital role in tissue-specific regulation of energy metabolism in mice. The DYRK/SIRT signaling axis will likely provide new insights into gene environment interactions that affect systemic energy metabolism, tissue homeostasis, and animal stress responses. Published 7. This article is a U.S. Government work and is in the public domain in the USA

14 Published online: March, 7 Tissue-specific SIRT phosphorylation Jing Lu et al Materials and Methods Animal experiments SIRT TAKI and mice were generated with standard mouse knock-in technology. Threonine of SIRT was targeted and replaced with alanine (TAKI) or glutamate () in C7BL/6 embryonic stem cells at Taconic Biosciences, Inc. The resulting TAKI,, and their littermates on the C7BL/6 background were housed in individualized ventilated cages (Tecniplast, Exton, PA) with a combination of autoclaved nesting material (Nestlet, Ancare Corp., Bellmore, NY and Crink-l Nest, The Andersons, Inc., Maumee, OH) and housed on hardwood bedding (Sani-chips, PJ Murphy, Montville, NJ, USA). Mice were maintained on a :-h light: dark cycle at. C and relative humidity of 4 6%. Mice were provided ad libitum autoclaved rodent diet (NIH, Harlan Laboratories, Madison, WI, USA) and deionized water treated by reverse osmosis. Mice were negative for mouse hepatitis virus, Sendai virus, pneumonia virus of mice, mouse parvovirus and, epizootic diarrhea of infant mice, mouse norovirus, Mycoplasma pulmonis, Helicobacter spp., and endo- and ectoparasites upon receipt, and no pathogens were detected in sentinel mice during this study. All the experiments were carried out on TAKI, mice and their gender- and age-matched littermates. Animal numbers in each group were chosen to achieve.-fold difference with 8% of power. No randomization method was used, and the investigators were not blinded to the group allocation. Fat and lean body mass were determined by Bruker LF9 minispec at the age of 9 months. High-fat Western diet (D79B, Research Diets) was used to feed mice aged months for weeks. Tissues were harvested after 4-h food withdrawal, starting at the beginning of the daytime cycle (fed), or after 6-h food withdrawal, starting at the end of daytime cycle (fasted). Tissue lipids were extracted as described []. Respiratory exchange ratio was measured using the Labmaster system (TSE systems). All animal procedures were reviewed and approved by National Institute of Environmental Health Sciences Animal Care and Use Committee. All animals were housed, cared for, and used in compliance with the Guide for the Care and Use of Laboratory Animals and housed and used in an Association for the Assessment and Accreditation of Laboratory Animal Care, International (AAALAC) Program. Histological analysis Liver samples were embedded with paraffin and stained with hematoxylin and eosin for morphological study. Differentiated adipocytes were collected at different time of differentiation and stained with Oil Red O as described [4]. Biochemical analysis Commercially available reagents or kits were used to measured serum lipids as listed: triglycerides and free glycerol reagent (Sigma), non-esterified fatty acid (NEFA) and cholesterol (Wako Pure Chemical Industries). Serum b-hydroxybutyrate was determined with Stanbio reagents (Stanbio). Liver lipids were extracted as described [], and liver triglycerides, NEFA, and cholesterol were quantified by above commercially available kits. Cell culture Based on a previously described method [], MEF cells were prepared from E4. embryos isolated from pregnant heterozygous (or TAKI) females mated with heterozygous (or TAKI) males. Isolated, TAKI, and MEF cells were further verified by PCR. MEF cells were maintained in Dulbecco s modified Eagle s medium (high glucose), supplemented with % fetal bovine serum (HyClone). To make immortalized MEF cells, lg of SV4 T antigen vector was transfected into 6 cells with Lipofectamine (Invitrogen). Transfected MEF cells were then split / at least five times to get immortalized cells. Protein phosphorylation, acetylation, and Western blot analysis To detect Thr phosphorylation level of SIRT in the mice, tissues were collected and homogenized in Nonidet P-4 buffer ( mm Tris HCl, ph 8., mm NaCl, % Nonidet P-4) containing complete protease inhibitors (Roche Applied Science). Western blot analysis was used to detect Thr-phosphorylated SIRT and total SIRT. When p was used as the acetylation substrate of SIRT,, TAKI, and their littermate MEF cells were treated with either. lg/ml of adriamycin (MP Biomedicals) or PBS (solvent control) for 8 h, with lm of trichostatin A (TSA, Sigma) and lm of MG- (Calbiochem) treatment during the last h. When NF-jB was used as the acetylation substrate of SIRT,, TAKI, and their littermate MEF cells were treated with either ng/ml of TNFa (R&D), lg/ml of Escherichia coli O:B4 lipopolysaccharide (LPS, Sigma) or PBS (solvent control) for min, also with nm of TSA. Cell lysates were prepared with SDS lysis buffer and resolved by SDS PAGE. Western blot analysis was used to detect acetylated substrates and total substrates. The acetylation levels of b-catenin were analyzed by immunoprecipitation of b-catenin followed by immunoblotting with an antiacetyl-k antibody. All the Western blot images were captured with Odyssey Infrared Imaging System (Li-Cor Biosciences). Antibodies used were listed as following: acetylated p (Millipore 6-96), total p (Santa Cruz Biotechnology sc-64), acetylated NF-jB (GeneTex GTX8696), total NF-jB (Santa Cruz Biotechnology sc-7), total SIRT (Sigma S96), b-catenin (GeneTex GTX4), GATA (Santa Cruz Biotechnology, sc-99), acetyl-lysine (Cell Signaling Technology #944). Rabbit anti-p-sirt(t) antiserum was generated and validated as described []. Please also see source data files for validation of these antibodies. Chromatin immunoprecipitation (ChIP) analysis Chromatin immunoprecipitation (ChIP) analysis was performed essentially as described by the manufacturer, EMD Millipore (Upstate Biotechnology). Briefly, and primary MEFs, or white adipose tissues from and mice were fixed with % paraformaldehyde in PBS at room temperature for min, minced, 4 Published 7. This article is a U.S. Government work and is in the public domain in the USA