Sirtuin1-regulated lysine acetylation of p66Shc governsdiabetes-induced vascular oxidative stress andendothelial dysfunctionSantosh Kumara,b,1, Young-Rae Kima,b, Ajit Vikrama,b, Asma Naqvic, Qiuxia Lia,b, Modar Kassana,b, Vikas Kumard,Markus M. Bachschmidd, Julia S. Jacobsa,b, Ajay Kumarc, and Kaikobad Irania,b,1

aDivision of Cardiovascular Medicine, Department of Internal Medicine, University of Iowa Carver College of Medicine, Iowa City, IA 52242; bAbboudCardiovascular Research Center, University of Iowa, Iowa City, IA 52242; cCardiovascular Institute, University of Pittsburgh, Pittsburgh, PA 15213; anddVascular Biology Section, Cardiovascular Proteomics Center, Boston University School of Medicine, Boston, MA 02118

Edited by Marc Montminy, The Salk Institute for Biological Studies, La Jolla, CA, and approved December 27, 2016 (received for review August 23, 2016)

The 66-kDa Src homology 2 domain-containing protein (p66Shc) isa master regulator of reactive oxygen species (ROS). It is expressed inmany tissues where it contributes to organ dysfunction by promotingoxidative stress. In the vasculature, p66Shc-induced ROS engendersendothelial dysfunction. Here we show that p66Shc is a direct targetof the Sirtuin1 lysine deacetylase (Sirt1), and Sirt1-regulated acetyla-tion of p66Shc governs its capacity to induce ROS. Using diabetes as anoxidative stimulus, we demonstrate that p66Shc is acetylated underhigh glucose conditions and is deacetylated by Sirt1 on lysine 81. Highglucose-stimulated lysine acetylation of p66Shc facilitates its phos-phorylation on serine 36 and translocation to the mitochondria, whereit promotes hydrogen peroxide production. Endothelium-specific trans-genic and global knockin mice expressing p66Shc that is not acetylat-able on lysine 81 are protected from diabetic oxidative stress andvascular endothelial dysfunction. These findings show that p66Shc is atarget of Sirt1, uncover a unique Sirt1-regulated lysine acetylation-dependent mechanism that governs the oxidative function of p66Shc,and demonstrate the importance of p66Shc lysine acetylation in vas-cular oxidative stress and diabetic vascular pathophysiology.

p66Shc | sirt1 | lysine acetylation | diabetes | oxidative stress

The 66-kDa Src homology 2 domain-containing protein(p66Shc) belongs to ShcA family of adaptor proteins. It is

unique in this family because unlike the p46 and p52 isoforms, itimpairs growth factor signaling and promotes oxidative stress (1).p66Shc contributes to aging (2), obesity (3, 4), atherosclerosis(5), and diabetes and aging-related vascular dysfunction (6, 7) inmice. In response to oxidative stimuli, p66Shc gets phosphory-lated at serine 36 (S36) and translocates to mitochondria, whereit produces reactive oxygen species (ROS) by oxidizing cytochromeC (8, 9). p66Shc is also phosphorylated on other residues that in-crease its half-life (10). In addition, p66Shc activates PKCBII,which in turn phosphorylates p66Shc, creating a positive feed-back loop (11). The importance of p66Shc in human disease issupported by evidence that its expression increases in patholo-gies such as diabetes and atherosclerosis (12, 13).Sirt1 belongs to the class III NAD+-dependent histone

deacetylases (HDACs). In lower organisms, it mediates lon-gevity in response to caloric restriction (14). In addition tohistones, it deacetylates many nonhistone proteins such as p53,FOXO, PGC1-alpha, LXR, and e-NOS (15–19). Sirt1 andp66Shc have opposing effects on vascular function (20). Unlikep66Shc, Sirt1 mitigates diabetes-induced vascular dysfunction(21). Moreover, down-regulation of Sirt1 in diabetes leads toepigenetic up-regulation of p66Shc (22). However, whetherSirt1 directly targets p66Shc for lysine deacetylation andwhether dynamic lysine acetylation of p66Shc governs its oxi-dative function are not known. Here we show that acetylationof lysine 81 in p66Shc is obligatory for diabetic vascular dys-function, and Sirt1 antagonizes this acetylation, thereby sup-pressing p66Shc-mediated oxidative stress.

Results and DiscussionSirt1 Deacetylates p66Shc on Lysine 81. We first determinedwhether the acetylation status of p66Shc is dynamic and gov-erned by Sirt1. Knockdown of Sirt1 using siRNA increased lysineacetylation of ectopically expressed p66Shc in human embryonickidney-293 (HEK 293) cells (SI Appendix, Fig. S1A) and led tohyperacetylation of endogenous p66Shc in human umbilical veinendothelial cells (HUVECs) (Fig. 1A and SI Appendix, Fig. S1B).Next, we investigated whether Sirt1 and p66Shc associate witheach other. Endogenous p66Shc in HUVECs coprecipitated withendogenous Sirt1 in HUVECs (Fig. 1B), and immunoprecipita-tion of overexpressed p66Shc in HEK 293 cells pulled downoverexpressed Sirt1 (SI Appendix, Fig. S1C).It is important to note that knockdown of Sirt1 also increased

lysine acetylation of p46 and p52Shc (SI Appendix, Fig. S1B),suggesting that there may be common lysine residues in all threeisoforms that are deacetylated by Sirt1. However, we focused ourattention on lysine residues in the N-terminal collagen homology2 (CH2) domain of p66Shc (2), which is not shared by p46 andp52Shc, as it is this CH2 domain that confers upon p66Shc itsunique oxidative function. There are three conserved lysine resi-dues in the CH2 domain at positions 7, 9, and 81 (SI Appendix,Fig. S2). To determine which, if any, of these three lysines is acet-ylated and targeted by Sirt1 for deacetylation, we first performedacetylation–deacetylation assays followed by mass spectrometry

Significance

Many oxidative stimuli engage the 66-kDa Src homology 2 domain-containing protein (p66Shc) to induce reactive oxygen species(ROS). ROS regulated by p66Shc promotes aging and contributesto cancer, diabetes, obesity, cardiomyopathy, and atherosclerosis.Here we identify a fundamental mechanism that controls p66Shcand p66Shc-regulated ROS. We show that p66Shc is lysine acety-lated when cells are faced with an oxidative stimulus (diabetes),and lysine acetylation of p66Shc is obligatory for p66Shc-inducedROS. In addition, lysine-acetylated p66Shc is deacetylated by theSirtuin1 lysine deacetylase, and Sirt1-mediated deacetylation ofp66Shc curtails ROS production. This intersection between p66Shcand Sirtuin1 adds a dimension to how p66Shc is regulated bycertain stimuli and how Sirtuin1 suppresses oxidative stress pro-moted by such stimuli.

Author contributions: S.K., M.M.B., and K.I. designed research; S.K., Y.-R.K., A.V., A.N., Q.L.,M.K., V.K., J.S.J., and A.K. performed research; S.K., Y.-R.K., A.V., and M.M.B. analyzed data;and S.K. and K.I. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence may be addressed. Email: kaikobad-irani@uiowa.edu orsantosh-kumar@uiowa.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1614112114/-/DCSupplemental.

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on recombinant CH2. His-tagged CH2 was acetylated in vitrowith p300 acetyltransferase followed by deacetylation by Sirt1.Immunoblotting showed that p300 induced lysine acetylation ofCH2, which was reversed by Sirt1 (Fig. 1C, Inset). CH2 acety-lated by p300 and deacetylated by Sirt1 was then subjected tomass spectrometry. Lysine 81 (K81) was identified as the mostabundantly acetylated and deacetylated residue (Fig. 1C and SIAppendix, Fig. S3 A and B). We developed an antibody againstK81-acetylated p66Shc (acetyl-p66ShcK81) and verified that itdetects only p300-acetylated and not nonacetylated recombinantCH2 (SI Appendix, Fig. S4A). Moreover, using this antibody, we

verified that Sirt1 deacetylates K81 in full-length p66Shc ex-pressed in HEK 293 cells (SI Appendix, Fig. S4B). Further, usingthis antibody, we showed that knockdown of Sirt1 in HUVECsengenders hyperacetylation of K81 in endogenous p66Shc (SIAppendix, Fig. S4C). To further verify that manipulation of Sirt1and p300 hyperacetylates K81 and that the acetyl-p66ShcK81antibody has specificity for Ac-K81, we created a p66Shc mutantthat is nonacetylatable on K81 (K81R). Knockdown of Sirt1 inHUVECs or overexpression of p300 in HEK 293 cells increasedacetylation on K81 in p66ShcWT but not p66ShcK81R (Fig. 1Dand SI Appendix, Fig. S4 D and E).

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Fig. 1. Sirt1 inhibits oxidative function of p66Shc by deacetylating it on lysine 81. (A) Immunoblot for acetylated p66Shc with Sirt1 knockdown in HUVECs.(B) Coimmunoprecipitation for endogenous Sirt1 and p66Shc in HUVECs. (C) Tandemmass spectrometry of recombinant CH2 domain of p66Shc acetylated in vitroby p300. Acetylated peptide shown has m/z 554.96. (Inset) Acetyl-lysine immunoblot of p300-acetylated and Sirt1-deacetylated CH2 domain used for massspectrometry. (D and E) Immunoblots for acetylp66ShcK81 (D) and phospho-p66ShcS36 (E) in HUVECs with Sirt1 knockdown. (F and G) Immunofluorescence foracetyl-p66ShcK81 (F) and phospho-p66ShcS36 (G) in aortas of mice with conditional deletion of endothelial Sirt1 (e-SIRT1KO) and wild-type mice. Arrow indicatesendothelial layer. (H) Quantification of endothelial acetyl-p66ShcK81 and phospho-p66ShcS36 from F and G. *P < 0.05, **P < 0.01; n = 3; Student’s t test.(I) Quantification of H2O2 (DCF fluorescence) in HUVECs expressing p66ShcWT or p66ShcK81R and treated with Sirt1 inhibitor NAM. ***P < 0.001; n = 3–7;Student’s t test. Data represent mean ± SEM. Immunoblots are representative of at least three independent experiments. vWF, von Willebrand factor.

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Deficiency of Sirt1 Promotes S36 Phosphorylation of p66Shc andp66Shc-Induced ROS via K81 Acetylation. There is precedent for lysineacetylation facilitating serine phosphorylation (23). Phosphorylationof p66Shc on S36 is essential for p66Shc-mediated ROS pro-duction (2). We therefore asked if phosphorylation of p66Shc

on S36 is dependent on acetylation of K81. Sirt1 knockdown inHUVECs increased S36 phosphorylation of p66Shc (SI Ap-pendix, Fig. S5A) and increased ROS (H2O2) levels in bothHUVECs and HEK 293 cells (SI Appendix, Fig. S5 B and C). Sirt1knockdown did not stimulate S36 phosphorylation of p66ShcK81R

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Fig. 2. Endothelial p66Shc is acetylated on lysine 81 by high glucose and in diabetes and promotes high glucose-induced mitochondrial oxidative stress. (A and B)Immunofluorescence for acetyly-p66ShcK81 (A) and quantification of acetyl-p66ShcK81 in aortic endothelium of STZ-induced diabetic mice. Magnified images areshown in Inset. Arrow indicates an endothelial layer. ***P < 0.001; n = 4–7; Student’s t test. (C and D) Immunoblot for high glucose-stimulated acetyl-p66ShcK81and phospho-p66ShcS36 in HUVECs expressing p66ShcWT or p66ShcK81R (C) and densitometric quantification of immunoblots (D). **P < 0.01, ***P < 0.001; n = 3;Student’s t test. (E and F) DCF fluorescence images (E) and quantification of fluorescence (F) of whole HUVECs expressing p66ShcWT or p66ShcK81R and incu-bated with high glucose. (G) Quantification of DCF fluorescence in mitochondria isolated from HUVECs expressing p66ShcWT or p66ShcK81R and incubated withhigh glucose. **P < 0.01, ***P < 0.001; n = 3–7; Student’s t test. (H and I) Immunoblots for p66Shc in mitochondrial and whole-cell lysates of HUVECs expressingp66ShcWT or p66ShcK81R and incubated with high glucose (H) and their densitometric quantification (I). **P < 0.01; n = 5; Student’s t test. Data represent means± SEM. STZ, streptozotocin. Immunoblots are representative of at least three independent experiments.

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expressed in either HEK 293 cells or HUVECs (Fig. 1E and SIAppendix, Fig. S5 D and E). In addition, both K81 acetylation andS36 phosphorylation were increased in the aortic endothelium ofmice with conditional deletion of endothelial Sirt1 (e-Sirt1 KO)(Fig. 1 F–H).We then examined the role of Sirt1-regulated K81 acetylation

in p66Shc-induced ROS production. Inhibition of Sirt1 with nic-otinamide (NAM) increased S36 phosphorylation in cells ex-pressing p66ShcWT but not in cells expressing p66ShcK81R (SIAppendix, Fig. S5 F and G). Further, expression of p66ShcWT,but not p66ShcK81R, amplified NAM-stimulated H2O2 inHUVECs (Fig. 1I). These findings underscore that inhibition ofSirt1 stimulates p66Shc-mediated ROS production via K81acetylation.

High Glucose-Stimulated K81 Acetylation Promotes MitochondrialTransport of p66Shc and p66Shc-Mediated Mitochondrial OxidativeStress. Diabetes promotes vascular oxidative stress via p66Shc(6). We evaluated the role of K81 acetylation in p66Shc-mediateddiabetic oxidative stress in the vascular endothelium. We firstexamined the acetylation status of vascular p66Shc in streptozo-tocin (STZ)-induced diabetic mice. STZ-induced diabetes led tohyperacetylation of endothelial p66Shc on K81 (Fig. 2 A and B). Asimilar increase in K81 acetylation was observed in HUVECsincubated in high glucose-containing medium (Fig. 2 C and D).High glucose also stimulated S36 phosphorylation of p66Shc inHUVECs (Fig. 2 C and D). Importantly, high glucose failed toinduce K81 acetylation or S36 phosphorylation of p66ShcK81R.Thus, K81 is acetylated in endothelial cells by high glucose in vitroand diabetes in vivo and facilitates S36 phosphorylation.We then evaluated the role of K81 acetylation in high glucose-

induced ROS mediated by p66Shc. High glucose-stimulatedH2O2 was blunted in HUVECs expressing p66ShcK81R com-pared with those expressing p66ShcWT (Fig. 2 E and F and SIAppendix, Fig. S6). Because of the role of p66Shc in mitochon-drial oxidative stress, we next asked if K81 acetylation is oblig-atory for high glucose-stimulated mitochondrial ROS mediatedby p66Shc. High glucose-stimulated H2O2 in isolated mito-chondria was significantly blunted in HUVECs expressingp66ShcK81R compared with those expressing p66ShcWT (Fig.2G). Thus, cells expressing p66ShcK81R are protected from highglucose-stimulated mitochondrial oxidative stress.In response to oxidative stimuli, a fraction of p66Shc trans-

locates to the mitochondrial intermembrane space in mito-chondria, where it oxidizes cytochrome c, resulting in oxidativestress and mitochondrial depolarization (8, 9). Given the im-portance of K81 acetylation in high glucose-stimulated ROS, weasked if K81 acetylation is also important for mitochondrialtranslocation of p66Shc. Incubation of HUVECs with high glu-cose led to an increase in both endogenous and overexpressedp66ShcWT in crude mitochondrial fraction (SI Appendix, Fig. S7A and B and Fig. 2 H and I). In contrast to p66ShcWT,p66ShcK81R did not accumulate in the mitochondria in re-sponse to high glucose (Fig. 2 H and I). These findings suggestthat K81 acetylation plays an important part in high glucose-stimulated mitochondrial translocation of p66Shc.

K81 Acetylation of p66Shc Promotes Diabetic Vascular EndothelialDysfunction. Diabetic vascular dysfunction is, in part, mediatedby p66Shc (6). To determine if K81 acetylation is required forp66Shc-mediated diabetic vascular dysfunction, we first expressedp66ShcK81R using a recombinant adenovirus (Ad-p66ShcK81R) inmouse aortas ex vivo (SI Appendix, Fig. S8A). Compared with ex-pression of p66ShcWT (Ad-p66ShcWT), expression of p66ShcK81Rdid not result in impairment of endothelium-dependent vaso-relaxation (SI Appendix, Fig. S8B). Endothelium-independentvascular relaxation was not different between mouse aortasexpressing p66ShcWT and p66ShcK81R (SI Appendix, Fig. S8C).

We also examined the effect of expressing p66ShcK81R in aortasof db/db diabetic mice that have impaired endothelium-dependentvasorelaxation. Expression of p66ShcK81R rescued endothelium-dependent vasorelaxation, whereas expression of p66ShcWTworsened it (SI Appendix, Fig. S8D and E). However, endothelium-independent relaxation was not different between db/db aortasexpressing p66ShcWT and p66ShcK81R (SI Appendix, Fig. S8F).To explore the in vivo role of p66Shc K81 acetylation in diabetic

vascular dysfunction, we generated a transgenic mouse with endo-thelium-specific expression of p66ShcK81R (henceforth callede-p66ShcK81R) (SI Appendix, Fig. S9A). These mice are viable andhealthy. Diabetes was induced by a single bolus injection of STZ (SIAppendix, Fig. S9B). Aortas of e-p66ShcK81R mice had improvedendothelium-dependent relaxation both under nondiabetic anddiabetic conditions, compared with their wild-type nontransgeniclittermate controls (Fig. 3A). Moreover, endothelium-specific oxi-dative stress (measured as 8-hydroxy deoxyguanosine, 8-OHdG)(Fig. 3 B and C) and acetylation of p66Shc on K81 in the endo-thelium were diminished in diabetic e-p66ShcK81R transgenic micecompared with diabetic wild-type nontransgenic littermates (SIAppendix, Fig. S9 C and D).To confirm the role of K81 acetylation in diabetic vascular dys-

function, we generated mice with global knockin of p66ShcK81R(SI Appendix, Fig. S10). These mice are fertile, viable, and healthy.There was no difference in STZ-induced hyperglycemia betweenp66ShcK81R knockin and wild-type mice (SI Appendix, Fig. S11).Although basal endothelium-dependent vasorelaxation was simi-lar in p66ShcK81R knockin and wild-type mice, p66ShcK81Rknockin mice were protected from STZ-induced impairmentof endothelium-dependent vasorelaxation (Fig. 3D). In addition,bioavailable vascular nitric oxide was higher in p66ShcK81Rknockin mice compared with wild-type mice in both the diabeticand nondiabetic states (Fig. 3E). Further, p66ShcK81R knockinmice were protected from STZ-induced vascular oxidative stress(Fig. 3 F and G).By showing that the oxidative function of p66Shc is governed

by lysine acetylation and acetylated p66Shc is a substrate forSirt1, this work uncovers dynamic lysine acetylation as anotherrheostat for the complex posttranslational regulation of p66Shc.The intersection between lysine acetylation and serine phos-phorylation is not unique to p66Shc. Lysine acetylation promotesprotein kinase B-mediated phosphorylation of Foxo1 (23),whereas phosphorylation of Beclin1 is required for its sub-sequent lysine acetylation (24). Interdependence between serinephosphorylation and lysine acetylation may be explained onstructural grounds of these posttranslational modifications (25).While our data indicate that K81 acetylation is required for

high glucose/diabetes-induced S36 phosphorylation, further studiesshowed that this requirement is not universally applicable for alloxidative stimuli. This was borne out in studies examining the effectof vascular endothelial growth factor (VEGF) on S36 phosphory-lation in p66ShcWT and p66ShcK81R. Although basal S36 phos-phorylation in serum-starved HUVECs was lower in p66ShcK81R,VEGF induced S36 phosphorylation to a similar extent in bothp66ShcWT and p66ShcK81R, without changing K81 acetylation(SI Appendix, Fig. S12). This stimulus-specific reliance of S36phosphorylation on K81 acetylation (present in high glucose/diabetes; absent in VEGF) could be explained by selective engage-ment of specific kinases by different oxidative stimuli, as phosphor-ylation of S36 is promiscuously induced by multiple kinases (26–28).An alternative explanation is that dependence of S36 phosphory-lation on K81 acetylation may be determined by whether thestimulus affects Sirt1 expression. Diabetes down-regulates en-dothelial Sirt1 (SI Appendix, Fig. S13), whereas some other ox-idative stimuli (such as VEGF) may not.Observations from clinical and preclinical studies suggest that

glycemic control alone is not sufficient to prevent diabetic com-plications and persistent oxidative stress could be responsible for

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this hyperglycemic memory (29, 30). One proposition that hasbeen forwarded as responsible for persistent vascular oxidativestress in diabetes is the epigenetic up-regulation of p66Shc. In thisregard, it has been shown that Sirt1 suppresses p66Shc expressionepigenetically, and genetic deletion of p66Shc protects mice againstdiabetic endothelial dysfunction and vascular hyperglycemic mem-ory (11, 22). Distinct from this finding, our work identifies an al-ternative mechanism by which Sirt1 regulates p66Shc—through

direct lysine deacetylation. Although acetylation of p66Shc is in-dependent from Sirt1-mediated epigenetic regulation of p66Shc,our data suggest some functional interplay between the two. This isborne out by the finding that suppression of K81 acetylation ofp66Shc in diabetic mice partially rescued vascular Sirt1 expression(SI Appendix, Fig. S13). Thus, p66Shc acetylation feeds back toinhibit Sirt1 expression, which in turn may also lead to up-regulationof p66Shc. Given that Sirt1 expression is governed by oxidative

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Fig. 3. Acetylation of p66Shc on lysine 81 mediates diabetic vascular oxidative stress and endothelial dysfunction. (A) Endothelium-dependent vaso-relaxation of aortas from nondiabetic and STZ-induced diabetic wild-type mice and transgenic mice with endothelial expression of p66ShcK81R(e-p66ShcK81R). ###P < 0.001 vs. wild type; *P < 0.05, ***P < 0.001 vs. wild-type STZ. Wild type (n = 12), e-p66ShcK81R (n = 18), wild-type STZ (n = 20), ande-p66ShcK81R STZ (n = 8). (B) Immunofluorescence for oxidative stress (8-OHdG; oxidative DNA adduct) in aortic sections of nondiabetic and STZ-induced di-abetic wild-type and e-p66ShcK81R mice. (C) Quantification of endothelial 8-OHdG in B. vWF, von Willebrand factor. ***P < 0.001; n = 5–8; Student’s t test. (D)Endothelium-dependent vasorelaxation of aortas of control (nondiabetic) and STZ-induced diabetic wild-type and p66ShcK81R knockin mice. #P < 0.05, ###P <0.001 vs. wild type. Wild type, n = 14; p66ShcK81R, n = 13; wild-type STZ, n = 15; and p66ShcK81R STZ, n = 21. (E) Nitric oxide bioavailability in aortas of controland STZ-induced diabetic p66ShcK81R knockin and wild-type mice. #P < 0.05, ##P < 0.01 vs. wild type; **P < 0.01 vs. wild-type STZ. Wild type, n = 14; p66ShcK81R,n = 13; wild-type STZ, n = 15; and p66ShcK81R STZ, n = 21. (F) Immunofluorescence for 8-OHdG in aortic sections of control and STZ-induced diabetic wild-typeand p66ShcK81R knockin mice. (G) Quantification of endothelial 8-OHdG in F. Arrow indicates endothelial layer. ***P < 0.001; n = 8–10; Student’s t test. 8-OHdG,8 hydroxy-deoxyguanosine; ACh, acetylcholine; PE, phenylephrine. n, number of aortic rings. All vascular reactivity data were analyzed by two-way ANOVAfollowed by Tukey’s post hoc analysis. Data represent mean ± SEM.

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stress (31), acetylated p66Shc may down-regulate Sirt1 by pro-moting vascular ROS production (SI Appendix, Fig. S14).In conclusion, these findings identify p66Shc as a target for

Sirt1 and show that Sirt1-mediated deacetylation of p66Shc has avital part in determining tissue vascular oxidative stress and en-dothelial dysfunction in diabetes. Although the studies were re-stricted to diabetes/high glucose as the oxidative stimulus and tovascular cells and tissue, the molecular mechanism by whichSirt1-regulated lysine acetylation of p66Shc governs ROS mayalso be operative in other oxidant-driven pathophysiology.

MethodsDetailed methods are described in SI Appendix.

Study Approval. All experimental animal procedures were approved by theInstitutional Animal Care and Use Committee at the University of Iowa, Iowa

City, and conformed to the Guide for the Care and Use of Laboratory Ani-mals published by the National Institutes of Health (32).

Statistics. All data met assumptions of the statistical test, and all statisticalanalysis was performed using GraphPad prism 6 unless specified. Data rep-resent means ± SEM of at least three independent assays unless otherwisestated. Significance of difference between two groups was determined us-ing two-tailed independent sample Student’s t test. All of the vascular re-activity data were analyzed by two-way ANOVA followed by Tukey’s posthoc analysis. Results were considered significant if P values were ≤ 0.05.

ACKNOWLEDGMENTS. We thank T. Finkel and L. Terada for the p66Shcconstructs and M. Joiner for CoxIV and Mitomix antibodies. K.I. wassupported by the University of Iowa Endowed Professorship in CardiovascularMedicine and by US Department of Veterans Affairs Grant 1I01BX002940;Q.L. was supported by NIH Grant T32 HL007344; and M.K. was supportedby NIH Grant T32 HL007121.

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Kumar et al. PNAS | February 14, 2017 | vol. 114 | no. 7 | 1719

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